Oligonucleotide analogues having modified intersubunit linkages and/or terminal groups

ABSTRACT

Oligonucleotide analogues comprising modified intersubunit linkages and/or modified 3′ and/or 5′-end groups are provided. The disclosed compounds are useful for the treatment of diseases where inhibition of protein expression or correction of aberrant mRNA splice products produces beneficial therapeutic effects.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit under 35 U.S.C. §119(e) ofU.S. Provisional Patent Application No. 61/349,783 filed on May 28,2010; U.S. Provisional Patent Application No. 61/361,878 filed on Jul.6, 2010 and U.S. Provisional Patent Application No. 61/386,428 filed onSep. 24, 2010, each of which are incorporated herein by reference intheir entireties.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 120178_(—)487C1_SEQUENCE_LISTING.txt. The textfile is about 18 KB, was created on Jun. 5, 2014, and is being submittedelectronically.

BACKGROUND

1. Technical Field

The present invention is generally related to oligonucleotide compounds(oligomers) useful as antisense compounds, and more particularly tooligomer compounds comprising modified intersubunit linkages and/orterminal groups, and the use of such oligomer compounds in antisenseapplications.

2. Description of the Related Art

Antisense oligomers are generally designed to bind to DNA or RNA ofdisease-causing proteins to prevent the production of such proteins.Requirements for successful implementation of antisense therapeuticsinclude (a) stability in vivo, (b) sufficient membrane permeability andcellular uptake, and (c) a good balance of binding affinity and sequencespecificity. Many oligonucleotide analogues have been developed in whichthe phosphodiester linkages of native DNA are replaced by other linkagesthat are resistant to nuclease degradation (see, e.g., Barawkar, D. A.et al., Proc. Nat'l Acad. Sci. USA 95(19):11047-52 (1998); Linkletter,B. A. et al., Nucleic Acids Res. 29(11):2370-6 (2001); Micklefield, J.,Curr, Med, Chem, 8(10):1157-79 (2001)). Antisense oligonucleotideshaving other various backbone modifications have also been prepared(Crooke, S. T., Antisense Drug Technology: Principles, Strategies, andApplications, New York, Marcel Dekker (2001); Micklefield, J., Curr,Med, Chem, 8(10):1157-79 (2001); Crooke, S. T., Antisense DrugTechnology, Boca Raton, CRC Press (2008)). In addition, oligonucleotideshave been modified by peptide conjugation in order to enhance cellularuptake (Moulton, H. M. et al., Bioconjug Chem 15(2):290-9 (2004);Nelson, M. H. et al., Bioconjug. Chem. 16(4):959-66 (2005); Moulton, H.M. et al., Biochim Biophys Acta (2010)).

The performance of such nucleic acid analogues as antisense or antigenedrugs has been hampered by certain characteristics of the variousanalogues. For example, analogues with negatively charged linkages,including phosphorothioate-linked analogues, suffer from considerableelectrostatic repulsion between the negative charges of the oligomer andthe DNA or RNA target. The phosphorothioates also exhibit non-specificbinding to other cellular components such as proteins. These attributeslimit the therapeutic effectiveness of antisense oligomers comprised ofnative RNA, native DNA, and negatively charged analogues (Crooke, S. T.,Antisense Drug Technology: Principles, Strategies, and Applications, NewYork, Marcel Dekker (2001); Crooke, S. T., Antisense Drug Technology,Boca Raton, CRC Press (2008)). The nonionic methylphosphonate-linkedoligonucleotide analogues can be transported into cells by passivediffusion and/or fluid phase endocytosis, but their use is hampered bystereoisomeric complexity and poor solubility (Crooke, S. T., AntisenseDrug Technology: Principles, Strategies, and Applications, New York,Marcel Dekker (2001); Micklefield, J., Curr, Med, Chem, 8(10):1157-79(2001)).

Several groups have reported the synthesis of positively chargedoligonucleotides (Bailey, C. P. et al. Nucleic Acids Res. 26(21):4860-7(1998); Micklefield, J., Curr, Med, Chem, 8(10):1157-79 (2001); Egli, M.et al., Biochemistry 44(25):9045-57 (2005)). For example, a class ofguanidinium linked nucleosides (designated DNG), formed by replacementof the phosphate linkages in DNA and RNA by achiral guanidino groups,has been reported (Dempcy, R. O. et al., Proc. Nat'l Acad. Sci. USA91(17):7864-8 (1994); Dempcy, R. O. et al., Proc. Nat'l Acad. Sci. USA93(9):4326-30 (1996); Barawkar, D. A. et al., Proc. Nat'l Acad. Sci. USA95(19):11047-52 (1998); Linkletter, B. A. et al., Nucleic Acids Res.29(11):2370-6 (2001)). Oligomers linked with positively chargedmethylated thiourea linkages have also been reported (Arya, D. P. etal., Proc. Nat'l Acad. Sci USA 96(8): 4384-9 (1999)). Replacement ofsome of these linkages with neutral urea linkages has been reported toreduce the tendency of such positively charged oligomers towardsnon-sequence-specific binding (Linkletter, B. A. et al., Bioorg. Med.Chem. 8(8):1893-901 (2000)). Morpholino oligomers containing(1-piperazino) phosphinylideneoxy and(1-(4-(ω-guanidino-alkanoyl))-piperazino) phosphinylideneoxy linkageshave been described previously (see e.g., WO2008036127).

Although significant progress has been made, there remains a need in theart for oligonucleotide analogues with improved antisense or antigeneperformance. Such improved antisense or antigene performance includes;stronger affinity for DNA and RNA without compromising sequenceselectivity; improved pharmacokinetics and tissue distribution; improvedcellular delivery and reliable and controllable in vivo distribution.

BRIEF SUMMARY

Compounds of the present invention address these issues and provideimprovements over existing antisense molecules in the art. Modificationof the intersubunit linkages and/or conjugation of terminal moieties tothe 5′ and/or 3′ terminus of an oligonucleotide analogue, for example amorpholino oligonucleotide, results in an antisense oligomer havingsuperior properties. For example, in certain embodiments the disclosedoligomers have enhanced cell delivery, potency, and/or tissuedistribution compared to other oligonucleotide analogues and/or can beeffectively delivered to the target organs. These superior propertiesgive rise to favorable therapeutic indices, reduced clinical dosing, andlower cost of goods.

In one embodiment, the present disclosure provides an oligomercomprising a backbone, the backbone comprising a sequence of morpholinoring structures joined by intersubunit linkages, the intersubunitlinkages joining a 3′-end of one morpholino ring structure to a 5′-endof an adjacent morpholino ring structure, wherein each morpholino ringstructure is bound to a base-pairing moiety, such that the oligomer canbind in a sequence-specific manner to a target nucleic acid, wherein theintersubunit linkages have the following general structure (I):

or a salt or isomer thereof, and wherein each of the intersubunitlinkages (I) are independently linkage (A) or linkage (B):

wherein for linkage (A):

-   -   W is, at each occurrence, independently S or O;    -   X is, at each occurrence, independently —N(CH₃)₂, —NR¹R², —OR³        or;

-   -   Y is, at each occurrence, independently O or —NR²,    -   R¹ is, at each occurrence, independently hydrogen or methyl;    -   R² is, at each occurrence, independently hydrogen or -LNR⁴R⁵R⁷;    -   R³ is, at each occurrence, independently hydrogen or C₁-C₆        alkyl;    -   R⁴ is, at each occurrence, independently hydrogen, methyl,        —C(═NH)NH₂, —Z-L-NHC(═NH)NH₂ or —[C(O)CHR′NH]_(m)H, where Z is        carbonyl (C(O)) or a direct bond, R′ is a side chain of a        naturally occurring amino acid or a one- or two-carbon homolog        thereof, and m is 1 to 6;    -   R⁵ is, at each occurrence, independently hydrogen, methyl or an        electron pair;    -   R⁶ is, at each occurrence, independently hydrogen or methyl;    -   R⁷ is, at each occurrence, independently hydrogen C₁-C₆ alkyl or        C₁-C₆ alkoxyalkyl;    -   L is an optional linker up to 18 atoms in length comprising        alkyl, alkoxy or alkylamino groups, or combinations thereof; and

wherein for linkage (B):

-   -   W is, at each occurrence, independently S or O;    -   X is, at each occurrence, independently —NR⁸R⁹ or —OR³; and    -   Y is, at each occurrence, independently O or —NR¹⁰,    -   R⁸ is, at each occurrence, independently hydrogen or C₂-C₁₂        alkyl;    -   R⁹ is, at each occurrence, independently hydrogen, C₁-C₁₂ alkyl,        C₁-C₁₂ aralkyl or aryl;    -   R¹⁰ is, at each occurrence, independently hydrogen, C₁-C₁₂ alkyl        or -LNR⁴R⁵R⁷;    -   wherein R⁸ and R⁹ may join to form a 5-18 membered mono or        bicyclic heterocycle or R⁸, R⁹ or R³ may join with R¹⁰ to form a        5-7 membered heterocycle, and wherein when X is 4-piparazino, X        has the following structure (III):

wherein:

-   -   R¹¹ is, at each occurrence, independently C₂-C₁₂ alkyl, C₁-C₁₂        aminoalkyl, C₁-C₁₂ alkylcarbonyl, aryl, heteroaryl or        heterocyclyl; and    -   R is, at each occurrence, independently an electron pair,        hydrogen or C₁-C₁₂ alkyl; and    -   R¹² is, at each occurrence, independently, hydrogen, C₁-C₁₂        alkyl, C₁-C₁₂ aminoalkyl, —NH₂, —CONH₂, —NR¹³R¹⁴, —NR¹³R¹⁴R¹⁵,        C₁-C₁₂ alkylcarbonyl, oxo, —CN, trifluoromethyl, amidyl,        amidinyl, amidinylalkyl, amidinylalkylcarbonyl guanidinyl,        guanidinylalkyl, guanidinylalkylcarbonyl, cholate, deoxycholate,        aryl, heteroaryl, heterocycle, —SR¹³ or C₁-C₁₂ alkoxy, wherein        R¹³, R¹⁴ and R¹⁵ are, at each occurrence, independently C₁-C₁₂        alkyl; and    -   wherein at least one of the intersubunit linkages is linkage        (B).

In another embodiment the present disclosure provides an oligomercomprising modified terminal groups, for example in one embodiment thedisclosure provides an oligomer comprising a backbone, the backbonecomprising a sequence of morpholino ring structures joined byintersubunit linkages of type (A), (B), or combinations thereof, whereineach morpholino ring structure supports a base-pairing moiety, such thatthe oligomer compound can bind in a sequence-specific manner to a targetnucleic acid, and wherein the oligomer comprises a 3′ terminus, a 5′terminus and has the following structure (XVII):

or a salt or isomer thereof, and

wherein for linkage (A):

-   -   W is, at each occurrence, independently S or O;    -   X is, at each occurrence, independently —N(CH₃)₂, —NR¹R², —OR³        or;

-   -   Y is, at each occurrence, independently O or —NR²,    -   R¹ is, at each occurrence, independently hydrogen or methyl;    -   R² is, at each occurrence, independently hydrogen or -LNR⁴R⁵R⁷;    -   R³ is, at each occurrence, independently hydrogen or C₁-C₆        alkyl;    -   R⁴ is, at each occurrence, independently hydrogen, methyl,        —C(═NH)NH₂, —Z-L-NHC(═NH)NH₂ or —[C(O)CHR′NH]_(m)H, where Z is        carbonyl (C(O)) or a direct bond, R′ is a side chain of a        naturally occurring amino acid or a one- or two-carbon homolog        thereof, and m is 1 to 6;    -   R⁵ is, at each occurrence, independently hydrogen, methyl or an        electron pair;    -   R⁶ is, at each occurrence, independently hydrogen or methyl;    -   R⁷ is, at each occurrence, independently hydrogen C₁-C₆ alkyl or        C₁-C₆ alkoxyalkyl;    -   L is an optional linker up to 18 atoms in length comprising        alkyl, alkoxy or alkylamino groups, or combinations thereof; and

wherein for linkage (B):

-   -   W is, at each occurrence, independently S or O;    -   X is, at each occurrence, independently —NR⁸R⁹ or —OR³; and    -   Y is, at each occurrence, independently O or —NR¹⁰,    -   R⁸ is, at each occurrence, independently hydrogen or C₂-C₁₂        alkyl;    -   R⁹ is, at each occurrence, independently hydrogen, C₁-C₁₂ alkyl,        C₁-C₁₂ aralkyl or aryl;    -   R¹⁰ is, at each occurrence, independently hydrogen, C₁-C₁₂ alkyl        or -LNR⁴R⁵R⁷;    -   wherein R⁸ and R⁹ may join to form a 5-18 membered mono or        bicyclic heterocycle or R⁸, R⁹ or R³ may join with R¹⁰ to form a        5-7 membered heterocycle, and wherein when X is 4-piparazino, X        has the following structure (III):

wherein:

-   -   R¹⁰ is, at each occurrence, independently C₂-C₁₂ alkyl, C₁-C₁₂        aminoalkyl, C₁-C₁₂ alkylcarbonyl, aryl, heteroaryl or        heterocyclyl; and    -   R¹¹ is, at each occurrence, independently an electron pair,        hydrogen or C₁-C₁₂ alkyl;    -   R¹² is, at each occurrence, independently, hydrogen, C₁-C₁₂        alkyl, C₁-C₁₂ aminoalkyl, —NH₂, —CONH₂, —NR¹³R¹⁴, —NR¹³R¹⁴R¹⁵,        C₁₂ alkylcarbonyl, —CN, trifluoromethyl, amidyl, amidinyl,        amidinylalkyl, amidinylalkylcarbonyl, guanidinyl,        guanidinylalkyl, guanidinylalkylcarbonyl, cholate, deoxycholate,        aryl, heteroaryl, heterocycle, —SR¹³ or C₁-C₁₂ alkoxy, wherein        R¹³, R¹⁴ and R¹⁵ are, at each occurrence, independently C₁-C₁₂        alkyl; and    -   R¹⁷ is, at each occurrence, independently absent, hydrogen or        C₁-C₆ alkyl;    -   R¹⁸ and R¹⁹ are, at each occurrence, independently absent,        hydrogen, a cell-penetrating peptide, a natural or non-natural        amino acid, C₂-C₃₀ alkylcarbonyl —C(═O)OR²¹ or R²⁰;    -   R²⁰ is, at each occurrence, independently guanidinyl,        heterocyclyl, C₁-C₃₀ alkyl, C₃-C₈ cycloalkyl; C₆-C₃₀ aryl,        C₇-C₃₀ aralkyl, C₃-C₃₀ alkylcarbonyl, C₃-C₈ cycloalkylcarbonyl,        C₃-C₈ cycloalkylalkylcarbonyl, C₇-C₃₀ arylcarbonyl, C₇-C₃₀        aralkylcarbonyl, C₂-C₃₀ alkyloxycarbonyl, C₃-C₈        cycloalkyloxycarbonyl, C₇-C₃₀ aryloxycarbonyl, C₈-C₃₀        aralkyloxycarbonyl, or —P(═O)(R²²)₂;    -   R²¹ is C₁-C₃₀ alkyl comprising one or more oxygen or hydroxyl        moieties or combinations thereof;    -   each R²² is independently C⁶-C¹² aryloxy;    -   B is a base-pairing moiety;    -   L¹ is an optional linker up to 18 atoms in length comprising        bonds selected from alkyl, hydroxyl, alkoxy, alkylamino, amide,        ester, disulfide, carbonyl, carbamate, phosphorodiamidate,        phosphoroamidate, phosphorothioate, piperazine and        phosphodiester;

x is an integer of 0 or greater; and

-   -   wherein at least one of R¹⁸ or R¹⁹ is R²⁰ and provided that both        of R¹⁷ and R¹⁸ are not absent.

In another embodiment, the present disclosure provides a method ofinhibiting production of a protein, the method comprising exposing anucleic acid encoding the protein to an oligomer of the presentdisclosure.

In another embodiment, the disclosure is directed to a method oftreating a disease in a subject, the method comprising administering atherapeutically effective amount of an oligomer. Methods of making theoligomers and methods for their use are also provided.

These and other aspects of the invention will be apparent upon referenceto the following detailed description. To this end, various referencesare set forth herein which describe in more detail certain backgroundinformation, procedures, compounds and/or compositions, and are eachhereby incorporated by reference in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows an exemplary morpholino oligomer structure comprising aphosphorodiamidate linkage.

FIG. 1B shows a morpholino oligomer as in FIG. 1A, but where thebackbone linkages comprise one piperazino phosphorodiamidate linkage.

FIG. 1C shows a conjugate of an arginine-rich peptide and an antisenseoligomer.

FIGS. 1D-G show the repeating subunit segment of exemplary morpholinooligonucleotides, designated 1D through 1G.

FIG. 2 depicts exemplary intersubunit linkages linked to a morpholino—Tmoiety.

FIG. 3 is a reaction scheme showing preparation of a linker forsolid-phase synthesis.

FIG. 4 demonstrates preparation of a solid support for oligomersynthesis.

FIG. 5 shows exon skipping activity of representative oligomers.

FIG. 6 is a bar graph showing exon skipping in the mdx mouse model.

FIGS. 7A-7C provides results of treatment of transgenic eGFP mice withexemplary oligomers.

FIG. 8 shows reduction in viral M2 protein levels from cells treatedwith exemplary oligomers.

FIG. 9 shows antiviral activity and weight loss in mice treated withexemplary oligomers.

FIG. 10 provides body weight data of mice treated with exemplaryoligomers.

FIG. 11 is eGFP splice-correction activity data in various tissues frommice treated with exemplary oligomers compared to PMO and PMO⁺oligomers.

FIG. 12 shows a subset of eGFP splice-correction activity data invarious tissues from mice treated with exemplary oligomers compared toPMO and PMO⁺ oligomers.

DETAILED DESCRIPTION I. Definitions

In the following description, certain specific details are set forth inorder to provide a thorough understanding of various embodiments.However, one skilled in the art will understand that the invention maybe practiced without these details. In other instances, well-knownstructures have not been shown or described in detail to avoidunnecessarily obscuring descriptions of the embodiments. Unless thecontext requires otherwise, throughout the specification and claimswhich follow, the word “comprise” and variations thereof, such as,“comprises” and “comprising” are to be construed in an open, inclusivesense, that is, as “including, but not limited to.” Further, headingsprovided herein are for convenience only and do not interpret the scopeor meaning of the claimed invention.

Reference throughout this specification to “one embodiment” or “anembodiment” means that a particular feature, structure or characteristicdescribed in connection with the embodiment is included in at least oneembodiment. Thus, the appearances of the phrases “in one embodiment” or“in an embodiment” in various places throughout this specification arenot necessarily all referring to the same embodiment. Furthermore, theparticular features, structures, or characteristics may be combined inany suitable manner in one or more embodiments. Also, as used in thisspecification and the appended claims, the singular forms “a,” “an,” and“the” include plural referents unless the content clearly dictatesotherwise. It should also be noted that the term “or” is generallyemployed in its sense including “and/or” unless the content clearlydictates otherwise.

The terms below, as used herein, have the following meanings, unlessindicated otherwise:

“Amino” refers to the —NH₂ radical.

“Cyano” or “nitrile” refers to the —CN radical.

“Hydroxy” or “hydroxyl” refers to the —OH radical.

“Imino” refers to the ═NH substituent.

“Guanidinyl” refers to the —NHC(═NH)NH₂ substituent.

“Amidinyl” refers to the —C(═NH)NH₂ substituent.

“Nitro” refers to the —NO₂ radical.

“Oxo” refers to the ═O substituent.

“Thioxo” refers to the ═S substituent.

“Cholate” refers to the following structure:

“Deoxycholate” refers to the following structure:

“Alkyl” refers to a straight or branched hydrocarbon chain radical whichis saturated or unsaturated (i.e., contains one or more double and/ortriple bonds), having from one to thirty carbon atoms, and which isattached to the rest of the molecule by a single bond. Alkyls comprisingany number of carbon atoms from 1 to 30 are included. An alkylcomprising up to 30 carbon atoms is referred to as a C₁-C₃₀ alkyl,likewise, for example, an alkyl comprising up to 12 carbon atoms is aC₁-C₁₂ alkyl. Alkyls (and other moieties defined herein) comprisingother numbers of carbon atoms are represented similarly. Alkyl groupsinclude, but are not limited to, C₁-C₃₀ alkyl, C₁-C₂₀ alkyl, C₁-C₁₅alkyl, C₁-C₁₀ alkyl, C₁-C₈ alkyl, C₁-C₆ alkyl, C₁-C₄ alkyl, C₁-C₃ alkyl,C₁-C₂ alkyl, C₂-C₈ alkyl, C₃-C₈ alkyl and C₄-C₈ alkyl. Representativealkyl groups include, but are not limited to, methyl, ethyl, n-propyl,1-methylethyl (iso-propyl), n-butyl, i-butyl, s-butyl, n-pentyl,1,1-dimethylethyl (t-butyl), 3-methylhexyl, 2-methylhexyl, ethenyl,prop-1-enyl, but-1-enyl, pent-1-enyl, penta-1,4-dienyl, ethynyl,propynyl, but-2-ynyl, but-3-ynyl, pentynyl, hexynyl, and the like.Unless stated otherwise specifically in the specification, an alkylgroup may be optionally substituted as described below.

“Alkylene” or “alkylene chain” refers to a straight or branched divalenthydrocarbon chain linking the rest of the molecule to a radical group.Alkylenes may be saturated or unsaturated (i.e., contains one or moredouble and/or triple bonds). Representative alkylenes include, but arenot limited to, C₁-C₁₂ alkylene, C₁-C₈ alkylene, C₁-C₆ alkylene, C₁-C₄alkylene, C₁-C₃ alkylene, C₁-C₂ alkylene, C₁ alkylene. Representativealkylene groups include, but are not limited to, methylene, ethylene,propylene, n-butylene, ethenylene, propenylene, n-butenylene,propynylene, n-butynylene, and the like. The alkylene chain is attachedto the rest of the molecule through a single or double bond and to theradical group through a single or double bond. The points of attachmentof the alkylene chain to the rest of the molecule and to the radicalgroup can be through one carbon or any two carbons within the chain.Unless stated otherwise specifically in the specification, an alkylenechain may be optionally substituted as described below.

“Alkoxy” refers to a radical of the formula —OR_(a) where R_(a) is analkyl radical as defined. Unless stated otherwise specifically in thespecification, an alkoxy group may be optionally substituted asdescribed below.

Alkoxyalkyl” refers to a radical of the formula —R_(b)OR_(a) where R_(a)is an alkyl radical as defined and where R_(b) is an alkylene radical asdefined. Unless stated otherwise specifically in the specification, analkoxyalkyl group may be optionally substituted as described below.

“Alkylcarbonyl” refers to a radical of the formula —C(═O)R_(a) whereR_(a) is an alkyl radical as defined above. Unless stated otherwisespecifically in the specification, an alkylcarbonyl group may beoptionally substituted as described below.

“Alkyloxycarbonyl” refers to a radical of the formula —C(═O)OR_(a) whereR_(a) is an alkyl radical as defined. Unless stated otherwisespecifically in the specification, an alkyloxycarbonyl group may beoptionally substituted as described below.

“Alkylamino” refers to a radical of the formula —NHR_(a) or —NR_(a)R_(a)where each R_(a) is, independently, an alkyl radical as defined above.Unless stated otherwise specifically in the specification, an alkylaminogroup may be optionally substituted as described below.

“Amidyl” refers to a radical of the formula —N(H)C(═O) R_(a) where R_(a)is an alkyl or aryl radical as defined herein. Unless stated otherwisespecifically in the specification, an amidyl group may be optionallysubstituted as described below.

“Amidinylalkyl” refers a radical of the formula —R_(b)—C(═NH)NH₂ whereR_(b) is an alkylene radical as defined above. Unless stated otherwisespecifically in the specification, an amidinylalkyl group may beoptionally substituted as described below.

“Amidinylalkylcarbonyl” refers a radical of the formula —C(═O)R_(b)—C(═NH)NH₂ where R_(b) is an alkylene radical as defined above. Unlessstated otherwise specifically in the specification, anamidinylalkylcarbonyl group may be optionally substituted as describedbelow.

“Aminoalkyl” refers to a radical of the formula —R_(b)—NR_(a)R_(a) whereR_(b) is an alkylene radical as defined above, and each R_(a) isindependently a hydrogen or an alkyl radical.

“Thioalkyl” refers to a radical of the formula —SR_(a) where R_(a) is analkyl radical as defined above. Unless stated otherwise specifically inthe specification, a thioalkyl group may be optionally substituted.

“Aryl” refers to a radical derived from a hydrocarbon ring systemcomprising hydrogen, 6 to 30 carbon atoms and at least one aromaticring. The aryl radical may be a monocyclic, bicyclic, tricyclic ortetracyclic ring system, which may include fused or bridged ringsystems. Aryl radicals include, but are not limited to, aryl radicalsderived from the hydrocarbon ring systems of aceanthrylene,acenaphthylene, acephenanthrylene, anthracene, azulene, benzene,chrysene, fluoranthene, fluorene, as-indacene, s-indacene, indane,indene, naphthalene, phenalene, phenanthrene, pleiadene, pyrene, andtriphenylene. Unless stated otherwise specifically in the specification,the term “aryl” or the prefix “ar-” (such as in “aralkyl”) is meant toinclude aryl radicals that are optionally substituted.

“Aralkyl” refers to a radical of the formula —R_(b)—R_(c) where R_(b) isan alkylene chain as defined above and R_(c) is one or more arylradicals as defined above, for example, benzyl, diphenylmethyl, trityland the like. Unless stated otherwise specifically in the specification,an aralkyl group may be optionally substituted.

“Arylcarbonyl” refers to a radical of the formula —C(═O)R_(c) whereR_(c) is one or more aryl radicals as defined above, for example,phenyl. Unless stated otherwise specifically in the specification, anarylcarbonyl group may be optionally substituted.

“Aryloxycarbonyl” refers to a radical of the formula —C(═O)OR_(c) whereR_(c) is one or more aryl radicals as defined above, for example,phenyl. Unless stated otherwise specifically in the specification, anaryloxycarbonyl group may be optionally substituted.

“Aralkylcarbonyl” refers to a radical of the formula —C(═O)R_(b)—R_(c)where R_(b) is an alkylene chain as defined above and R_(c) is one ormore aryl radicals as defined above, for example, phenyl. Unless statedotherwise specifically in the specification, an aralkylcarbonyl groupmay be optionally substituted.

“Aralkyloxycarbonyl” refers to a radical of the formula—C(═O)OR_(b)—R_(c) where R_(b) is an alkylene chain as defined above andR_(c) is one or more aryl radicals as defined above, for example,phenyl. Unless stated otherwise specifically in the specification, anaralkyloxycarbonyl group may be optionally substituted.

“Aryloxy” refers to a radical of the formula —OR_(c) where R_(c) is oneor more aryl radicals as defined above, for example, phenyl. Unlessstated otherwise specifically in the specification, an arylcarbonylgroup may be optionally substituted.

“Cycloalkyl” refers to a stable, non-aromatic, monocyclic or polycycliccarbocyclic ring, which may include fused or bridged ring systems, whichis saturated or unsaturated, and attached to the rest of the molecule bya single bond. Representative cycloalkyls include, but are not limitedto, cycloaklyls having from three to fifteen carbon atoms and from threeto eight carbon atoms, Monocyclic cycicoalkyl radicals include, forexample, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,and cyclooctyl. Polycyclic radicals include, for example, adamantyl,norbornyl, decalinyl, and 7,7-dimethyl-bicyclo[2.2.1]heptanyl. Unlessotherwise stated specifically in the specification, a cycloalkyl groupmay be optionally substituted.

“Cycloalkylalkyl” refers to a radical of the formula —R_(b)R_(d) whereR_(b) is an alkylene chain as defined above and R_(d) is a cycloalkylradical as defined above. Unless stated otherwise specifically in thespecification, a cycloalkylalkyl group may be optionally substituted.

“Cycloalkylcarbonyl” refers to a radical of the formula —C(═O)R_(d)where R_(d) is a cycloalkyl radical as defined above. Unless statedotherwise specifically in the specification, a cycloalkylcarbonyl groupmay be optionally substituted.

Cycloalkyloxycarbonyl” refers to a radical of the formula —C(═O)OR_(d)where R_(d) is a cycloalkyl radical as defined above. Unless statedotherwise specifically in the specification, a cycloalkyloxycarbonylgroup may be optionally substituted.

“Fused” refers to any ring structure described herein which is fused toan existing ring structure. When the fused ring is a heterocyclyl ringor a heteroaryl ring, any carbon atom on the existing ring structurewhich becomes part of the fused heterocyclyl ring or the fusedheteroaryl ring may be replaced with a nitrogen atom.

“Guanidinylalkyl” refers a radical of the formula —R_(b)—NHC(═NH)NH₂where R_(b) is an alkylene radical as defined above. Unless statedotherwise specifically in the specification, a guanidinylalkyl group maybe optionally substituted as described below.

“Guanidinylalkylcarbonyl” refers a radical of the formula—C(═O)R_(b)—NHC(═NH)NH₂ where R_(b) is an alkylene radical as definedabove. Unless stated otherwise specifically in the specification, aguanidinylalkylcarbonyl group may be optionally substituted as describedbelow.

“Halo” or “halogen” refers to bromo, chloro, fluoro or iodo.

“Haloalkyl” refers to an alkyl radical, as defined above, that issubstituted by one or more halo radicals, as defined above, e.g.,trifluoromethyl, difluoromethyl, fluoromethyl, trichloromethyl,2,2,2-trifluoroethyl, 1,2-difluoroethyl, 3-bromo-2-fluoropropyl,1,2-dibromoethyl, and the like. Unless stated otherwise specifically inthe specification, a haloalkyl group may be optionally substituted.

“Perhalo” or “perfluoro” refers to a moiety in which each hydrogen atomhas been replaced by a halo atom or fluorine atom, respectively.

“Heterocyclyl”, “heterocycle” or “heterocyclic ring” refers to a stable3- to 24-membered non-aromatic ring radical comprising 2 to 23 carbonatoms and from one to 8 heteroatoms selected from the group consistingof nitrogen, oxygen, phosphorous and sulfur. Unless stated otherwisespecifically in the specification, the heterocyclyl radical may be amonocyclic, bicyclic, tricyclic or tetracyclic ring system, which mayinclude fused or bridged ring systems; and the nitrogen, carbon orsulfur atoms in the heterocyclyl radical may be optionally oxidized; thenitrogen atom may be optionally quaternized; and the heterocyclylradical may be partially or fully saturated. Examples of suchheterocyclyl radicals include, but are not limited to, dioxolanyl,thienyl[1,3]dithianyl, decahydroisoquinolyl, imidazolinyl,imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl,octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl,2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl,piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl,thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl,thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl,1,1-dioxo-thiomorpholinyl, 12-crown-4, 15-crown-5, 18-crown-6,21-crown-7, aza-18-crown-6, diaza-18-crown-6, aza-21-crown-7, anddiaza-21-crown-7. Unless stated otherwise specifically in thespecification, a heterocyclyl group may be optionally substituted.

“Heteroaryl” refers to a 5- to 14-membered ring system radicalcomprising hydrogen atoms, one to thirteen carbon atoms, one to sixheteroatoms selected from the group consisting of nitrogen, oxygen,phosphorous and sulfur, and at least one aromatic ring. For purposes ofthis invention, the heteroaryl radical may be a monocyclic, bicyclic,tricyclic or tetracyclic ring system, which may include fused or bridgedring systems; and the nitrogen, carbon or sulfur atoms in the heteroarylradical may be optionally oxidized; the nitrogen atom may be optionallyquaternized. Examples include, but are not limited to, azepinyl,acridinyl, benzimidazolyl, benzothiazolyl, benzindolyl, benzodioxolyl,benzofuranyl, benzooxazolyl, benzothiazolyl, benzothiadiazolyl,benzo[b][1,4]dioxepinyl, 1,4-benzodioxanyl, benzonaphthofuranyl,benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl,benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl(benzothiophenyl), benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl,carbazolyl, cinnolinyl, dibenzofuranyl, dibenzothiophenyl, furanyl,furanonyl, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl,isoindolyl, indolinyl, isoindolinyl, isoquinolyl, indolizinyl,isoxazolyl, naphthyridinyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl,oxiranyl, 1-oxidopyridinyl, 1-oxidopyrimidinyl, 1-oxidopyrazinyl,1-oxidopyridazinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl,phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl,pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, quinazolinyl,quinoxalinyl, quinolinyl, quinuclidinyl, isoquinolinyl,tetrahydroquinolinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl,triazinyl, and thiophenyl (i.e., thienyl). Unless stated otherwisespecifically in the specification, a heteroaryl group may be optionallysubstituted.

All the above groups may be either substituted or unsubstituted. Theterm “substituted” as used herein means any of the above groups (i.e.,alkyl, alkylene, alkoxy, alkoxyalkyl, alkylcarbonyl, alkyloxycarbonyl,alkylamino, amidyl, amidinylalkyl, amidinylalkylcarbonyl, aminoalkyl,aryl, aralkyl, arylcarbonyl, aryloxycarbonyl, aralkylcarbonyl,aralkyloxycarbonyl, aryloxy, cycloalkyl, cycloalkylalkyl,cycloalkylcarbonyl, cycloalkylalkylcarbonyl, cycloalkyloxycarbonyl,guanidinylalkyl, guanidinylalkylcarbonyl, haloalkyl, heterocyclyl and/orheteroaryl), may be further functionalized wherein at least one hydrogenatom is replaced by a bond to a non-hydrogen atom substituent. Unlessstated specifically in the specification, a substituted group mayinclude one or more substituents selected from: oxo, —CO₂H, nitrile,nitro, —CONH₂, hydroxyl, thiooxy, alkyl, alkylene, alkoxy, alkoxyalkyl,alkylcarbonyl, alkyloxycarbonyl, aryl, aralkyl, arylcarbonyl,aryloxycarbonyl, aralkylcarbonyl, aralkyloxycarbonyl, aryloxy,cycloalkyl, cycloalkylalkyl, cycloalkylcarbonyl,cycloalkylalkylcarbonyl, cycloalkyloxycarbonyl, heterocyclyl,heteroaryl, dialkylamines, arylamines, alkylarylamines, diarylamines,N-oxides, imides, and enamines; a silicon atom in groups such astrialkylsilyl groups, dialkylarylsilyl groups, alkyldiarylsilyl groups,triarylsilyl groups, perfluoroalkyl or perfluoroalkoxy, for example,trifluoromethyl or trifluoromethoxy. “Substituted” also means any of theabove groups in which one or more hydrogen atoms are replaced by ahigher-order bond (e.g., a double- or triple-bond) to a heteroatom suchas oxygen in oxo, carbonyl, carboxyl, and ester groups; and nitrogen ingroups such as imines, oximes, hydrazones, and nitriles. For example,“substituted” includes any of the above groups in which one or morehydrogen atoms are replaced with —NR_(g)C(═O)NR_(g)R_(h),—NR_(g)C(═O)OR_(h), —NR_(g)SO₂R_(h), —OC(═O)NR_(g)R_(h), —OR_(g),—SR_(g), —SOR_(g), —SO₂R_(g), —OSO₂R_(g), —SO₂OR_(g), ═NSO₂R_(g), and—SO₂NR_(g)R_(h). “Substituted” also means any of the above groups inwhich one or more hydrogen atoms are replaced with —C(═O)R_(g),—C(═O)OR_(g), —CH₂SO₂R_(g), —CH₂SO₂NR_(g)R_(h), —SH, —SR_(g) or—SSR_(g). In the foregoing, R_(g) and R_(h) are the same or differentand independently hydrogen, alkyl, alkoxy, alkylamino, thioalkyl, aryl,aralkyl, cycloalkyl, cycloalkylalkyl, haloalkyl, heterocyclyl,N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl and/orheteroarylalkyl. In addition, each of the foregoing substituents mayalso be optionally substituted with one or more of the abovesubstituents. Furthermore, any of the above groups may be substituted toinclude one or more internal oxygen or sulfur atoms. For example, analkyl group may be substituted with one or more internal oxygen atoms toform an ether or polyether group. Similarly, an alkyl group may besubstituted with one or more internal sulfur atoms to form a thioether,disulfide, etc. Amidyl moieties may be substituted with up to 2 haloatoms, while other groups above may be substituted with one or more haloatoms. With the exception of alkyl groups, all other groups may also besubstituted with amino or monoalklyamino. With the exception of alkyland alkylcarbonyl groups, all other groups may also be substituted withguanidinyl or amidynyl. Optional substitutents for any of the abovegroups also include arylphosphoryl, for example —R_(a)P(Ar)₃ wherein Rais an alkylene and Ar is aryl moiety, for example phenyl.

The terms “antisense oligomer” or “antisense compound” are usedinterchangeably and refer to a sequence of subunits, each having a basecarried on a backbone subunit composed of ribose or other pentose sugaror morpholino group, and where the backbone groups are linked byintersubunit linkages that allow the bases in the compound to hybridizeto a target sequence in a nucleic acid (typically an RNA) byWatson-Crick base pairing, to form a nucleic acid:oligomer heteroduplexwithin the target sequence. The oligomer may have exact sequencecomplementarity to the target sequence or near complementarity. Suchantisense oligomers are designed to block or inhibit translation of themRNA containing the target sequence, and may be said to be “directed to”a sequence with which it hybridizes.

A “morpholino oligomer” or “PMO” refers to a polymeric molecule having abackbone which supports bases capable of hydrogen bonding to typicalpolynucleotides, wherein the polymer lacks a pentose sugar backbonemoiety, and more specifically a ribose backbone linked by phosphodiesterbonds which is typical of nucleotides and nucleosides, but insteadcontains a ring nitrogen with coupling through the ring nitrogen. Anexemplary“morpholino” oligomer comprises morpholino subunit structureslinked together by (thio)phosphoramidate or (thio)phosphorodiamidatelinkages, joining the morpholino nitrogen of one subunit to the 5′exocyclic carbon of an adjacent subunit, each subunit comprising apurine or pyrimidine base-pairing moiety effective to bind, bybase-specific hydrogen bonding, to a base in a polynucleotide.Morpholino oligomers (including antisense oligomers) are detailed, forexample, in U.S. Pat. Nos. 5,698,685; 5,217,866; 5,142,047; 5,034,506;5,166,315; 5,185,444; 5,521,063; 5,506,337 and pending U.S. patentapplication Ser. Nos. 12/271,036; 12/271,040; and PCT publication numberWO/2009/064471 all of which are incorporated herein by reference intheir entirety. Representative PMOs include PMOs wherein theintersubunit linkages are linkage (A1).

“PMO+” refers to phosphorodiamidate morpholino oligomers comprising anynumber of (1-piperazino)phosphinylideneoxy,(1-(4-(ω-guanidino-alkanoyl))-piperazino)phosphinylideneoxy linkages (A2and A3) that have been described previously (see e.g., PCT publicationWO/2008/036127 which is incorporated herein by reference in itsentirety.

“PMO-X” refers to phosphorodiamidate morpholino oligomers disclosedherein comprising at least one (B) linkage or at least one of thedisclosed terminal modifications.

A “phosphoramidate” group comprises phosphorus having three attachedoxygen atoms and one attached nitrogen atom, while a“phosphorodiamidate” group (see e.g., FIGS. 1D-E) comprises phosphorushaving two attached oxygen atoms and two attached nitrogen atoms. In theuncharged or the modified intersubunit linkages of the oligomersdescribed herein and co-pending U.S. Patent Application No. 61/349,783and Ser. No. 11/801,885, one nitrogen is always pendant to the backbonechain. The second nitrogen, in a phosphorodiamidate linkage, istypically the ring nitrogen in a morpholino ring structure.

“Thiophosphoramidate” or “thiophosphorodiamidate” linkages arephosphoramidate or phosphorodiamidate linkages, respectively, whereinone oxygen atom, typically the oxygen pendant to the backbone, isreplaced with sulfur.

“Intersubunit linkage” refers to the linkage connecting two morpholinosubunits, for example structure (I).

“Charged”, “uncharged”, “cationic” and “anionic” as used herein refer tothe predominant state of a chemical moiety at near-neutral pH, e.g.,about 6 to 8. For example, the term may refer to the predominant stateof the chemical moiety at physiological pH, that is, about 7.4.

“Lower alkyl” refers to an alkyl radical of one to six carbon atoms, asexemplified by methyl, ethyl, n-butyl, i-butyl, t-butyl, isoamyl,n-pentyl, and isopentyl. In certain embodiments, a “lower alkyl” grouphas one to four carbon atoms. In other embodiments a “lower alkyl” grouphas one to two carbon atoms; i.e. methyl or ethyl. Analogously, “loweralkenyl” refers to an alkenyl radical of two to six, preferably three orfour, carbon atoms, as exemplified by allyl and butenyl.

A “non-interfering” substituent is one that does not adversely affectthe ability of an antisense oligomer as described herein to bind to itsintended target. Such substituents include small and/or relativelynon-polar groups such as methyl, ethyl, methoxy, ethoxy, or fluoro.

An oligonucleotide or antisense oligomer “specifically hybridizes” to atarget polynucleotide if the oligomer hybridizes to the target underphysiological conditions, with a Tm greater than 37° C., greater than45° C., preferably at least 50° C., and typically 60° C.-80° C. orhigher. The “Tm” of an oligomer is the temperature at which 50%hybridizes to a complementary polynucleotide. Tm is determined understandard conditions in physiological saline, as described, for example,in Miyada et al., Methods Enzymol. 154:94-107 (1987). Such hybridizationmay occur with “near” or “substantial” complementary of the antisenseoligomer to the target sequence, as well as with exact complementarity.

Polynucleotides are described as “complementary” to one another whenhybridization occurs in an antiparallel configuration between twosingle-stranded polynucleotides. Complementarity (the degree that onepolynucleotide is complementary with another) is quantifiable in termsof the proportion of bases in opposing strands that are expected to formhydrogen bonds with each other, according to generally acceptedbase-pairing rules.

A first sequence is an “antisense sequence” with respect to a secondsequence if a polynucleotide whose sequence is the first sequencespecifically binds to, or specifically hybridizes with, the secondpolynucleotide sequence under physiological conditions.

The term “targeting sequence” is the sequence in the oligonucleotideanalog that is complementary (meaning, in addition, substantiallycomplementary) to the target sequence in the RNA genome. The entiresequence, or only a portion, of the analog compound may be complementaryto the target sequence. For example, in an analog having 20 bases, only12-14 may be targeting sequences. Typically, the targeting sequence isformed of contiguous bases in the analog, but may alternatively beformed of non-contiguous sequences that when placed together, e.g., fromopposite ends of the analog, constitute sequence that spans the targetsequence.

Target and targeting sequences are described as “complementary” to oneanother when hybridization occurs in an antiparallel configuration. Atargeting sequence may have “near” or “substantial” complementarity tothe target sequence and still function for the purpose of the presentlydescribed methods, that is, still be “complementary.” Preferably, theoligonucleotide analog compounds employed in the presently describedmethods have at most one mismatch with the target sequence out of 10nucleotides, and preferably at most one mismatch out of 20.Alternatively, the antisense oligomers employed have at least 90%sequence homology, and preferably at least 95% sequence homology, withthe exemplary targeting sequences as designated herein. For purposes ofcomplementary binding to an RNA target, and as discussed below, aguanine base may be complementary to either a cytosineor uracil RNAbase.

A “heteroduplex” refers to a duplex between an oligonculeotide analogand the complementary portion of a target RNA. A “nuclease-resistantheteroduplex” refers to a heteroduplex formed by the binding of anantisense oligomer to its complementary target, such that theheteroduplex is substantially resistant to in vivo degradation byintracellular and extracellular nucleases, such as RNAse H, which arecapable of cutting double-stranded RNA/RNA or RNA/DNA complexes.

An agent is “actively taken up by mammalian cells” when the agent canenter the cell by a mechanism other than passive diffusion across thecell membrane. The agent may be transported, for example, by “activetransport”, referring to transport of agents across a mammalian cellmembrane by e.g. an ATP-dependent transport mechanism, or by“facilitated transport”, referring to transport of antisense agentsacross the cell membrane by a transport mechanism that requires bindingof the agent to a transport protein, which then facilitates passage ofthe bound agent across the membrane.

The terms “modulating expression” and/or “antisense activity” refer tothe ability of an antisense oligomer to either enhance or, moretypically, reduce the expression of a given protein, by interfering withthe expression or translation of RNA. In the case of reduced proteinexpression, the antisense oligomer may directly block expression of agiven gene, or contribute to the accelerated breakdown of the RNAtranscribed from that gene. Morpholino oligomers as described herein arebelieved to act via the former (steric blocking) mechanism. Preferredantisense targets for steric blocking oligomers include the ATG startcodon region, splice sites, regions closely adjacent to splice sites,and 5′-untranslated region of mRNA, although other regions have beensuccessfully targeted using morpholino oligomers.

An “amino acid subunit” is preferably an α-amino acid residue(—CO—CHR—NH—); it may also be a β- or other amino acid residue (e.g.—CO—CH₂CHR—NH—), where R is an amino acid side chain.

The term “naturally occurring amino acid” refers to an amino acidpresent in proteins found in nature. The term “non-natural amino acids”refers to those amino acids not present in proteins found in nature;examples include beta-alanine (β-Ala) and 6-aminohexanoic acid (Ahx).

An “effective amount” or “therapeutically effective amount” refers to anamount of antisense oligomer administered to a mammalian subject, eitheras a single dose or as part of a series of doses, which is effective toproduce a desired therapeutic effect, typically by inhibitingtranslation of a selected target nucleic acid sequence.

“Treatment” of an individual (e.g. a mammal, such as a human) or a cellis any type of intervention used in an attempt to alter the naturalcourse of the individual or cell. Treatment includes, but is not limitedto, administration of a pharmaceutical composition, and may be performedeither prophylactically or subsequent to the initiation of a pathologicevent or contact with an etiologic agent.

II. Antisense Oligomers A. Oligomers with Modified Intersubunit Linkages

As noted above, one embodiment of the present disclosure is directed tooligomers comprising novel intersubunit linkages. In some embodiments,the oligomers have higher affinity for DNA and RNA than do thecorresponding unmodified oligomers and demonstrate improved celldelivery, potency, and/or tissue distribution properties compared tooligomers having other intersubunit linkages. In one embodiment, theoligomers comprise at least one intersubunit linkage of type (B) asdefined above. The oligomers may also comprise one or more intersubunitlinkages of type (A) as defined above. The structural features andproperties of the various linkage types and oligomers are described inmore detail in the following discussion.

1. Linkage (A)

Applicants have found that enhancement of antisense activity,biodistribution and/or other desirable properties can be optimized bypreparing oligomers having various intersubunit linkages. For example,the oligomers may optionally comprise one or more intersubunit linkagesof type (A), and in certain embodiments the oligomers comprise at leastone linkage of type (A). In some other embodiments each linkage of type(A) has the same structure. Linkages of type (A) may include linkagesdisclosed in co-owned U.S. Pat. No. 7,943,762 which is herebyincorporated by reference in its entirety. Linkage (A) has the followingstructure (I), wherein 3′ and 5′ indicate the point of attachment to the3′ and 5′ ends, respectively, of the morpholino ring (i.e., structure(i) discussed below):

or a salt or isomer thereof, wherein:

W is, at each occurrence, independently S or O;

X is, at each occurrence, independently —N(CH₃)₂, —NR¹R², —OR³ or;

Y is, at each occurrence, independently O or —NR²,

R¹ is, at each occurrence, independently hydrogen or methyl;

R² is, at each occurrence, independently hydrogen or -LNR⁴R⁵R⁷;

R³ is, at each occurrence, independently hydrogen or C₁-C₆ alkyl;

R⁴ is, at each occurrence, independently hydrogen, methyl, —C(═NH)NH₂,—Z-L-NHC(═NH)NH₂ or —[C(═O)CHR′NH]_(m)H, where Z is —C(═O)— or a directbond, R′ is a side chain of a naturally occurring amino acid or a one-or two-carbon homolog thereof, and m is 1 to 6;

R⁵ is, at each occurrence, independently hydrogen, methyl or an electronpair;

R⁶ is, at each occurrence, independently hydrogen or methyl;

R⁷ is, at each occurrence, independently hydrogen C₁-C₆ alkyl or C₁-C₆alkoxyalkyl; and

L is an optional linker up to 18 atoms in length comprising alkyl,alkoxy or alkylamino groups, or combinations thereof.

In some examples, the oligomer comprises at least one linkage of type(A). In some other embodiments, the oligomer includes at least twoconsecutive linkages of type (A). In further embodiments, at least 5% ofthe linkages in the oligomer are type (A); for example in someembodiments, 5%-95%, 10% to 90%, 10% to 50%, or 10% to 35% of thelinkages may be linkage type (A). In some specific embodiments, at leastone type (A) linkage is —N(CH₃)₂. In other embodiments, each linkage oftype (A) is —N(CH₃)₂. In other embodiments, at least one type (A)linkage is piperizin-1-yl, for example unsubstituted piperazin-1-yl(e.g., A2 or A3). In other embodiments, each linkage of type (A) ispiperizin-1-yl, for example unsubstituted piperazin-1-yl.

In some embodiments, W is, at each occurrence, independently S or O, andin certain embodiments W is O.

In some embodiments, X is, at each occurrence, independently —N(CH₃)₂,—NR¹R², —OR³. In some embodiments X is —N(CH₃)₂. In other aspects X is—NR¹R², and in other examples X is —OR³.

In some embodiments, R¹ is, at each occurrence, independently hydrogenor methyl. In some embodiments, R¹ is hydrogen. In other embodiments Xis methyl.

In some embodiments, R² is, at each occurrence, hydrogen. In otherembodiments R² is, at each occurrence, -LNR⁴R⁵R⁷. In some embodiments,R³ is, at each occurrence, independently hydrogen or C₁-C₆ alkyl. Inother embodiments, R³ is methyl. In yet other embodiments, R³ is ethyl.In some other embodiments, R³ is n-propyl or isopropyl. In some otherembodiments, R³ is C₄ alkyl. In other embodiments, R³ is C₅ alkyl. Insome embodiments, R³ is C₆ alkyl.

In certain embodiments, R⁴ is, at each occurrence, independentlyhydrogen. In other embodiments, R⁴ is methyl. In yet other embodiments,R⁴ is —C(═NH)NH₂, and in other embodiments, R⁴ is —Z-L-NHC(═NH)NH₂. Instill other embodiments, R⁴ is —[C(═O)CHR′NH]_(m)H. Z is —C(═O)— in oneembodiment and Z is a direct bond in another embodiment. R′ is a sidechain of a naturally occurring amino acid. In some embodiments R′ is aone- or two-carbon homolog of a side chain of a naturally occurringamino acid.

m is and integer from 1 to 6. m may be 1. m may be 2 m may be 3 m may be4 m may be 5 m may be 6

In some embodiments, R⁵ is, at each occurrence, independently hydrogen,methyl or an electron pair. In some embodiments, R⁵ is hydrogen. Inother embodiments, R⁵ is methyl. In yet other embodiments, R⁵ is anelectron pair.

In some embodiments, R⁶ is, at each occurrence, independently hydrogenor methyl. In some embodiments, R⁶ is hydrogen. In other embodiments, R⁶is methyl.

In other embodiments, R⁷ is, at each occurrence, independently hydrogenC₁-C₆ alkyl or C₂-C₆ alkoxyalkyl. In some embodiments R7 is hydrogen. Inother embodiments, R⁷ is C₁-C₆ alkyl. In yet other embodiments, R⁷ isC₂-C₆ alkoxyalkyl. In some embodiments, R⁷ is methyl. In otherembodiments, R⁷ is ethyl. In yet other embodiments, R⁷ is n-propyl orisopropyl. In some other embodiments, R⁷ is C₄ alkyl. In someembodiments, R⁷ is C₅ alkyl. In some embodiments, R⁷ is C₆ alkyl. In yetother embodiments, R⁷ is C₂ alkoxyalkyl. In some other embodiments, R⁷is C₃ alkoxyalkyl. In yet other embodiments, R⁷ is C₄ alkoxyalkyl. Insome embodiments, R⁷ is C₅ alkoxyalkyl. In other embodiments, R⁷ is C₆alkoxyalkyl.

The linker group L, as noted above, contains bonds in its backboneselected from alkyl (e.g. —CH2-CH2-), alkoxy (e.g., —C—O—C—), andalkylamino (e.g. —CH2-NH—), with the proviso that the terminal atoms inL (e.g., those adjacent to carbonyl or nitrogen) are carbon atoms.Although branched linkages (e.g. —CH2-CHCH3-) are possible, the linkeris generally unbranched. In one embodiment, the linker is a hydrocarbonlinker. Such a linker may have the structure (CH2)_(n)—, where n is1-12, preferably 2-8, and more preferably 2-6.

Oligomers having any number of linkage type (A) are provided. In someembodiments, the oligomer contains no linkages of type (A). In certainembodiments, 5, 10, 20, 30, 40, 50, 60, 70, 80 or 90 percent of thelinkages are linkage (A). In selected embodiments, 10 to 80, 20 to 80,20 to 60, 20 to 50, 20 to 40, or 20 to 35 percent of the linkages arelinkage (A).

2. Linkage (B)

In some embodiments, the oligomers comprise at least one linkage of type(B). For example the oligomers may comprise 1, 2, 3, 4, 5, 6 or morelinkages of type (B). The type (B) linkages may be adjacent or may beinterspersed throughout the oligomer. Linkage type (B) has the followingstructure (I):

or a salt or isomer thereof, wherein:

W is, at each occurrence, independently S or O;

X is, at each occurrence, independently —NR⁸R⁹ or —OR³; and

Y is, at each occurrence, independently O or —NR¹⁰,

R³ is, at each occurrence, independently hydrogen or C₁-C₆ alkyl;

R⁸ is, at each occurrence, independently hydrogen or C₂-C₁₂ alkyl;

R⁹ is, at each occurrence, independently hydrogen, C₁-C₁₂ alkyl, C₁-C₁₂aralkyl or aryl;

R¹⁰ at each occurrence, independently hydrogen, C₁-C₁₂ alkyl or-LNR⁴R⁵R⁷;

wherein R⁸ and R⁹ may join to form a 5-18 membered mono or bicyclicheterocycle or R⁸, R⁹ or R³ may join with R¹⁰ to form a 5-7 memberedheterocycle, and wherein when X is 4-piparazino, X has the followingstructure (III):

wherein:

R¹¹ is, at each occurrence, independently C₂-C₁₂ alkyl, C₁-C₁₂aminoalkyl, C₁-C₁₂ alkylcarbonyl, aryl, heteroaryl or heterocyclyl;

R is, at each occurrence, independently an electron pair, hydrogen orC₁-C₁₂ alkyl; and

R¹² is, at each occurrence, independently, hydrogen, C₁-C₁₂ alkyl,C₁-C₁₂ aminoalkyl, —NH₂, —CONH₂, —NR¹³R¹⁴, —NR¹³R¹⁴R¹⁵, C₁-C₁₂alkylcarbonyl, oxo, —CN, trifluoromethyl, amidyl, amidinyl,amidinylalkyl, amidinylalkylcarbonyl guanidinyl, guanidinylalkyl,guanidinylalkylcarbonyl, cholate, deoxycholate, aryl, heteroaryl,heterocycle, —SR¹³ or C₁-C₁₂ alkoxy, wherein R¹³, R¹⁴ and R¹⁵ are, ateach occurrence, independently C₁-C₁₂ alkyl.

In some examples, the oligomer comprises one linkage of type (B). Insome other embodiments, the oligomer comprises two inkages of type (B).In some other embodiments, the oligomer comprises three linkages of type(B). In some other embodiments, the oligomer comprises four linkages oftype (B). In still other embodiments, the linkages of type (B) areconsecutive (i.e., the type (B) linkages are adjacent to each other). Infurther embodiments, at least 5% of the linkages in the oligomer aretype (B); for example in some embodiments, 5%-95%, 10% to 90%, 10% to50%, or 10% to 35% of the linkages may be linkage type (B).

In other embodiments, R³ is, at each occurrence, independently hydrogenor C₁-C₆ alkyl. In yet other embodiments, R³ may be methyl. In someembodiments, R³ may be ethyl. In some other embodiments, R³ may ben-propyl or isopropyl. In yet other embodiments, R³ may be C₄ alkyl. Insome embodiments, R³ may be C₅ alkyl. In some embodiments, R³ may be C₆alkyl.

In some embodiments, R⁸ is, at each occurrence, independently hydrogenor C₂-C₁₂ alkyl. In some embodiments, R⁸ is hydrogen. In yet otherembodiments, R⁸ is ethyl. In some other embodiments, R⁸ is n-propyl orisopropyl. In some embodiments, R⁸ is C₄ alkyl. In yet otherembodiments, R⁸ is C₅ alkyl. In other embodiments, R⁸ is C₆ alkyl. Insome embodiments, R⁸ is C₇ alkyl. In yet other embodiments, R⁸ is C₈alkyl. In other embodiments, R⁸ is C₉ alkyl. In yet other embodiments,R⁸ is C₁₀ alkyl. In some other embodiments, R⁸ is C₁₁ alkyl. In yetother embodiments, R⁸ is C₁₂ alkyl. In some other embodiments, R⁸ isC₂-C₁₂ alkyl and the C₂-C₁₂ alkyl includes one or more double bonds(e.g., alkene), triple bonds (e.g., alkyne) or both. In someembodiments, R⁸ is unsubstituted C₂-C₁₂ alkyl.

In some embodiments, R⁹ is, at each occurrence, independently hydrogen,C₁-C₁₂ alkyl, C₁-C₁₂ aralkyl or aryl. In some embodiments, R⁹ ishydrogen. In yet other embodiments, R⁹ is C₁-C₁₂ alkyl. In otherembodiments, R⁹ is methyl. In yet other embodiments, R⁹ is ethyl. Insome other embodiments, R⁹ is n-propyl or isopropyl. In someembodiments, R⁹ is C₄ alkyl. In some embodiments, R⁹ is C₅ alkyl. In yetother embodiments, R⁹ is C₆ alkyl. In some other embodiments, R⁹ is C₇alkyl. In some embodiments, R⁹ is C₈ alkyl. In some embodiments, R⁹ isC₉ alkyl. In some other embodiments, R⁹ is C₁₀ alkyl. In some otherembodiments, R⁹ is C₁₁ alkyl. In yet other embodiments, R⁹ is C₁₂ alkyl.

In some other embodiments, R⁹ is C₁-C₁₂ aralkyl. For example, n someembodiments R⁹ is benzyl and the benzyl may be optionally substituted oneither the phenyl ring or the benzylic carbon. Substituents in thisregards include alkyl and alkoxy groups, for example methyl or methoxy.In some embodiments, the benzyl group is substituted with methyl at thebenzylic carbon. For example, in some embodiments, R⁹ has the followingstructure (XIV):

In other embodiments, R⁹ is aryl. For example, in some embodiments R⁹ isphenyl, and the phenyl may be optionally substituted. Substituents inthis regard substitutents include alkyl and alkoxy groups, for examplemethyl or methoxy. In other embodiments, R⁹ is phenyl and the phenylcomprises a crown ether moiety, for example a 12-18 membered crownether. In one embodiment the crown ether is 18 membered and may furthercomprise and additional phenyl moiety. For example, in one embodiment R⁹has one of the following structures (XV) or XVI):

In some embodiments, R¹⁰ is, at each occurrence, independently hydrogen,C₁-C₁₂ alkyl or -LNR⁴R⁵R⁷, wherein R⁴, R⁵ and R⁷ are as defined abovewith respect to linkage (A). In other embodiments, R¹⁰ is hydrogen. Inother embodiments, R¹⁰ is C₁-C₁₂ alkyl, and in other embodiments R¹⁰ is-LNR⁴R⁵R⁷. In some embodiments, R¹⁰ is methyl. In yet other embodiments,R¹⁰ is ethyl. In some embodiments, R¹⁰ is C₃ alkyl. In some embodiments,R¹⁰ is C₄ alkyl. In yet other embodiments, R¹⁰ is C₅ alkyl. In someother embodiments, R¹⁰ is C₆ alkyl. In other embodiments, R¹⁰ is C₇alkyl. In yet other embodiments, R¹⁰ is C₈ alkyl. In some embodiments,R¹⁰ is C₉ alkyl. In other embodiments, R¹⁰ is C₁₀ alkyl. In yet otherembodiments, R¹⁰ is C₁₁ alkyl. In some other embodiments, R¹⁰ is C₁₂alkyl.

In some embodiments, R⁸ and R⁹ join to form a 5-18 membered mono orbicyclic heterocycle. In some embodiments the heterocycle is a 5 or 6membered monocyclic heterocycle. For example, in some embodimentslinkage (B) has the following structure (IV):

In other embodiments, heterocycle is bicyclic, for example a 12-memberedbicyclic heterocycle. The heterocycle may be piperizinyl. Theheterocycle may be morpholino. The heterocycle may be piperidinyl. Theheterocycle may be decahydroisoquinoline. Representative heterocyclesinclude the following:

In some embodiments, R¹¹ is, at each occurrence, independently C₂-C₁₂alkyl, C₁-C₁₂ aminoalkyl, aryl, heteroaryl or heterocyclyl.

In some embodiments, R¹¹ is C₂-C₁₂ alkyl. In some embodiments, R¹¹ isethyl. In other embodiments, R¹¹ is C₃ alkyl. In yet other embodiments,R¹¹ is isopropyl. In some other embodiments, R¹¹ is C₄ alkyl. In otherembodiments, R¹¹ is C₅ alkyl. In some embodiments, R¹¹ is C₆ alkyl. Inother embodiments, R¹¹ is C₇ alkyl. In some embodiments, R¹¹ is C₈alkyl. In other embodiments, R¹¹ is C₉ alkyl. In yet other embodiments,R¹¹ is C₁₀ alkyl. In some other embodiments, R¹¹ is C₁₁ alkyl. In someembodiments, R¹¹ is C₁₂ alkyl.

In other embodiments, R¹¹ is C₁-C₁₂ aminoalkyl. In some embodiments, R¹¹is methylamino. In some embodiments, R¹¹ is ethylamino. In otherembodiments, R¹¹ is C₃ aminoalkyl. In yet other embodiments, R¹¹ is C₄aminoalkyl. In some other embodiments, R¹¹ is C₅ aminoalkyl. In otherembodiments, R¹¹ is C₆ aminoalkyl. In yet other embodiments, R¹¹ is C₇aminoalkyl. In some embodiments, R¹¹ is C₈ aminoalkyl. In otherembodiments, R¹¹ is C₉ aminoalkyl. In yet other embodiments, R¹¹ is C₁₀aminoalkyl. In some other embodiments, R¹¹ is C₁₁ aminoalkyl. In otherembodiments, R¹¹ is C₁₂ aminoalkyl.

In other embodiments, R¹¹ is C₁-C₁₂ alkylcarbonyl. In yet otherembodiments, R¹¹ is C₁ alkylcarbonyl. In other embodiments, R¹¹ is C₂alkylcarbonyl. In some embodiments, R¹¹ is C₃ alkylcarbonyl. In yetother embodiments, R¹¹ is C₄ alkylcarbonyl. In some embodiments, R¹¹ isC₅ alkylcarbonyl. In some other embodiments, R¹¹ is C₆ alkylcarbonyl. Inother embodiments, R¹¹ is C₇ alkylcarbonyl. In yet other embodiments,R¹¹ is C₈ alkylcarbonyl. In some embodiments, R¹¹ is C₉ alkylcarbonyl.In yet other embodiments, R¹¹ is C₁₀ alkylcarbonyl. In some otherembodiments, R¹¹ is C₁₁ alkylcarbonyl. In some embodiments, is C₁₂alkylcarbonyl. In yet other embodiments, R¹¹ is —C(═O)(CH₂)_(n)CO₂H,where n is 1 to 6. For example, in some embodiments, n is 1. In otherembodiments, n is 2. In yet other embodiments, n is 3. In some otherembodiments, n is 4. In yet other embodiments, n is 5. In otherembodiments, n is 6.

In other embodiments, R¹¹ is aryl. For example, in some embodiments, R¹¹is phenyl. In some embodiments, the phenyl is substituted, for examplewith a nitro group.

In other embodiments, R¹¹ is heteroaryl. For example, in someembodiments, R¹¹ is pyridinyl. In other embodiments, R¹¹ is pyrimidinyl.

In other embodiments, R¹¹ is heterocyclyl. For example, in someembodiments, R¹¹ is piperidinyl, for example piperidin-4-yl.

In some embodiments, R¹¹ is ethyl, isopropyl, piperidinyl, pyrimidinyl,cholate, deoxycholate, or —C(═O)(CH₂)_(n)CO₂H, where n is 1 to 6.

In some embodiments, R is an electron pair. In other embodiments, R ishydrogen, and in other embodiments R is C₁-C₁₂ alkyl. In someembodiments, R is methyl. In some embodiments, R is ethyl. In otherembodiments, R is C₃ alkyl. In yet other embodiments, R is isopropyl. Insome other embodiments, R is C₄ alkyl. In yet other embodiments, R is C₅alkyl. In some embodiments, R is C₆ alkyl. In other embodiments, R is C₇alkyl. In yet other embodiments, R is C₈ alkyl. In other embodiments, Ris C₉ alkyl. In some embodiments, R is C₁₀ alkyl. In yet otherembodiments, R is C₁₁ alkyl. In some embodiments, R is C₁₂ alkyl.

In some embodiments, R¹² is, at each occurrence, independently,hydrogen, C₁-C₁₂ alkyl, C₁-C₁₂ aminoalkyl, —NH₂, —CONH₂, —NR¹³R¹⁴,—NR¹³R¹⁴R¹⁵, oxo, —CN, trifluoromethyl, amidyl, amidinyl, amidinylalkyl,amidinylalkylcarbonyl guanidinyl, guanidinylalkyl,guanidinylalkylcarbonyl, cholate, deoxycholate, aryl, heteroaryl,heterocycle, —SR¹³ or C₁-C₁₂ alkoxy, wherein R¹³, R¹⁴ and R¹⁵ are, ateach occurrence, independently C₁-C₁₂ alkyl

In some embodiments, R¹² is hydrogen. In some embodiments, R¹² is C₁-C₁₂alkyl. In some embodiments, R¹² is C₁-C₁₂ aminoalkyl. In someembodiments, R¹² is —NH₂. In some embodiments, R¹² is —CONH₂. In someembodiments, R¹² is —NR¹³R¹⁴. In some embodiments, R¹² is —NR¹³R¹⁴R¹⁵.In some embodiments, R¹² is C₁-C₁₂ alkylcarbonyl. In some embodiments,R¹² is oxo. In some embodiments, R¹² is —CN. In some embodiments, R¹² istrifluoromethyl. In some embodiments, R¹² is amidyl. In someembodiments, R¹² is amidinyl. In some embodiments, R¹² is amidinylalkyl.In some embodiments, R¹² is amidinylalkylcarbonyl. In some embodiments,R¹² is guanidinyl, for example mono methylguanidynyl ordimethylguanidinyl. In some embodiments, R¹² is guanidinylalkyl. In someembodiments, R¹² is amidinylalkylcarbonyl. In some embodiments, R¹² ischolate. In some embodiments, R¹² is deoxycholate. In some embodiments,R¹² is aryl. In some embodiments, R¹² is heteroaryl. In someembodiments, R¹² is heterocycle. In some embodiments, R¹² is —SR¹³. Insome embodiments, R¹² is C₁-C₁₂ alkoxy. In some embodiments, R¹² isdimethyl amine.

In other embodiments, R¹² is methyl. In yet other embodiments, R¹² isethyl. In some embodiments, R¹² is C₃ alkyl. In some embodiments, R¹² isisopropyl. In some embodiments, R¹² is C₄ alkyl. In other embodiments,R¹² is C₅ alkyl. In yet other embodiments, R¹² is C₆ alkyl. In someother embodiments, R¹² is C₇ alkyl. In some embodiments, R¹² is C₈alkyl. In yet other embodiments, R¹² is C₉ alkyl. In some embodiments,R¹² is C₁₀ alkyl. In yet other embodiments, R¹² is C₁₁ alkyl. In otherembodiments, R¹² is C₁₂ alkyl. In yet other embodiments, the alkylmoiety is substituted with one or more oxygen atom to form an ethermoiety, for example a methoxymethyl moiety.

In some embodiments, R¹² is methylamino. In other embodiments, R¹² isethylamino. In yet other embodiments, R¹² is C₃ aminoalkyl. In someembodiments, R¹² is C₄ aminoalkyl. In yet other embodiments, R¹² is C₅aminoalkyl. In some other embodiments, R¹² is C₆ aminoalkyl. In someembodiments, R¹² is C₇ aminoalkyl. In some embodiments, R¹² is C₈aminoalkyl. In yet other embodiments, R¹² is C₉ aminoalkyl. In someother embodiments, R¹² is C₁₀ aminoalkyl. In yet other embodiments, R¹²is C₁₁ aminoalkyl. In other embodiments, R¹² is C₁₂ aminoalkyl. In someembodiments, the amino alkyl is a dimethylamino alkyl.

In yet other embodiments, R¹² is acetyl. In some other embodiments, R¹²is C₂ alkylcarbonyl. In some embodiments, R¹² is C₃ alkylcarbonyl. Inyet other embodiments, R¹² is C₄ alkylcarbonyl. In some embodiments, R¹²is C₅ alkylcarbonyl. In yet other embodiments, R¹² is C₆ alkylcarbonyl.In some other embodiments, R¹² is C₇ alkylcarbonyl. In some embodiments,R¹² is C₈ alkylcarbonyl. In yet other embodiments, R¹² is C₉alkylcarbonyl. In some other embodiments, R¹² is C₁₀ alkylcarbonyl. Insome embodiments, R¹² is C₁₁ alkylcarbonyl. In other embodiments, R¹² isC₁₂ alkylcarbonyl. The alkylcarbonyl is substituted with a carboxymoiety, for example the alkylcarbonyl is substituted to form a succinicacid moiety (i.e., a 3-carboxyalkylcarbonyl). In other embodiments, thealkylcarbonyl is substituted with a terminal —SH group.

In some embodiments, R¹² is amidyl. In some embodiments, the amidylcomprises an alkyl moiety which is further substituted, for example with—SH, carbamate, or combinations thereof. In other embodiments, theamidyl is substituted with an aryl moiety, for example phenyl. Incertain embodiments, R¹² may have the following structure (IX):

wherein R¹⁶ is, at each occurrence, independently hydrogen, C₁-C₁₂alkyl, C₁-C₁₂ alkoxy, —CN, aryl or heteroaryl.

In some embodiments, R¹² is methoxy. In other embodiments, R¹² isethoxy. In yet other embodiments, R¹² is C₃ alkoxy. In some embodiments,R¹² is C₄ alkoxy. In some embodiments, R¹² is C₅ alkoxy. In some otherembodiments, R¹² is C₆ alkoxy. In other embodiments, R¹² is C₇ alkoxy.In some other embodiments, R¹² is C₈ alkoxy. In some embodiments, R¹² isC₉ alkoxy. In other embodiments, R¹² is C₁₀ alkoxy. In some embodiments,R¹² is C₁₁ alkoxy. In yet other embodiments, R¹² is C₁₂ alkoxy.

In certain embodiments, R¹² is pyrrolidinyl, for examplepyrrolidin-1-yl. In other embodiments, R¹² is piperidinyl, for examplepiperidin-1-yl or piperidin-4-yl. In other embodiment, R¹² ismorpholino, for example morpholin-4-yl. In other embodiments, R¹² isphenyl, and in even further embodiments, the phenyl is substituted, forexample with a nitro group. In still other embodiments, R¹² ispyrimidinyl, for example pyrimidin-2-yl.

In other embodiments, R¹³, R¹⁴ and R¹⁵ are, at each occurrence,independently C₁-C₁₂ alkyl. In some embodiments, R¹³, R¹⁴ or R¹⁵ ismethyl. In yet other embodiments, R¹³, R¹⁴ or R¹⁵ is ethyl. In otherembodiments, R¹³, R¹⁴ or R¹⁵ is C₃ alkyl. In yet other embodiments, R¹³,R¹⁴ or R¹⁵ is isopropyl. In other embodiments, R¹³, R¹⁴ or R¹⁵ is C₄alkyl. In some embodiments, R¹³, R¹⁴ or R¹⁵ is C₅ alkyl. In some otherembodiments, R¹³, R¹⁴ or R¹⁵ is C₆ alkyl. In other embodiments, R¹³, R¹⁴or R¹⁵ is C₇ alkyl. In yet other embodiments, R¹³, R¹⁴ or R¹⁵ is C₈alkyl. In other embodiments, R¹³, R¹⁴ or R¹⁵ is C₉ alkyl. In someembodiments, R¹³, R¹⁴ or R¹⁵ is C₁₀ alkyl. In some embodiments, R¹³, R¹⁴or R¹⁵ is C₁₁ alkyl. In yet other embodiments, R¹³, R¹⁴ or R¹⁵ is C₁₂alkyl.

As noted above, in some embodiments, R¹² is amidyl substituted with anaryl moiety. In this regard, each occurrence of R¹⁶ may be the same ordifferent. In certain of these embodiments, R¹⁶ is hydrogen. In otherembodiments, R¹⁶ is —CN. In other embodiments, R¹⁶ is heteroaryl, forexample tretrazolyl. In certain other embodiments, R¹⁶ is methoxy. Inother embodiments, R¹⁶ is aryl, and the aryl is optionally substituted.Optional substitutents in this regard include: C₁-C₁₂ alkyl, C₁-C₁₂alkoxy, for example methoxy; trifluoromethoxy; halo, for example chloro;and trifluoromethyl.

In other embodiments, R¹⁶ is methyl. In yet other embodiments, R¹⁶ isethyl. In some embodiments, R¹⁶ is C₃ alkyl. In some other embodiments,R¹⁶ is isopropyl. In yet other embodiments, R¹⁶ is C₄ alkyl. In otherembodiments, R¹⁶ is C₅ alkyl. In yet other embodiments, R¹⁶ is C₆ alkyl.In some other embodiments, R¹⁶ is C₇ alkyl. In some embodiments, R¹⁶ isC₈ alkyl. In yet other embodiments, R¹⁶ is C₉ alkyl. In some otherembodiments, R¹⁶ is C₁₀ alkyl. In other embodiments, R¹⁶ is C₁₁ alkyl.In some other embodiments, R¹⁶ is C₁₂ alkyl.

In some embodiments, R¹⁶ is methoxy. In some embodiments, R¹⁶ is ethoxy.In yet other embodiments, R¹⁶ is C₃ alkoxy. In some other embodiments,R¹⁶ is C₄ alkoxy. In other embodiments, R¹⁶ is C₅ alkoxy. In some otherembodiments, R¹⁶ is C₆ alkoxy. In yet other embodiments, R¹⁶ is C₇alkoxy. In some other embodiments, R¹⁶ is C₈ alkoxy. In yet otherembodiments, R¹⁶ is C₉ alkoxy. In some other embodiments, R¹⁶ is C₁₀alkoxy. In some embodiments, R¹⁶ is C₁₁ alkoxy. In some otherembodiments, R¹⁶ is C₁₂ alkoxy.

In some other embodiments, R⁸ and R⁹ join to form a 12-18 membered crownether. For example, in some embodiments, the crown ether s 18 membered,and in other embodiments the crown ether is 15 membered. In certainembodiments, R⁸ and R⁹ join to form a heterocycle having one of thefollowing structures (X) or (XI):

In some embodiments, R⁸, R⁹ or R³ join with R¹⁰ to form a 5-7 memberedheterocycle. For example, in some embodiments, R³ joins with R¹⁰ to forma 5-7 membered heterocycle. In some embodiments, the heterocycle is5-membered. In other embodiments, the heterocycle is 6-membered. Inother embodiments, the heterocycle is 7-membered. In some embodiments,the heterocycle is represented by the following structure (XII):

wherein Z′ represents a 5-7 membered heterocycle. In certain embodimentsof structure (XI), R¹² is hydrogen at each occurrence. For example,linkage (B) may have one of the following structures (B1), (B2) or (B3):

In certain other embodiments, R¹² is C₁-C₁₂ alkylcarbonyl or amidylwhich is further substituted with an arylphosphoryl moiety, for examplea triphenyl phosporyl moiety. Examples of linkages having this structureinclude B56 and B55.

In certain embodiment, linkage (B) does not have any of the structuresA1-A5. Table 1 shows representative linkages of type (A) and (B).

TABLE 1 Representative Intersubunit Linkages No. Name Structure A1  PMO

A2  PMO⁺ (unprotonated form depicted)

A3  PMO⁺ (+)

A4  PMO^(mepip) (m+)

A5  PMO^(GUX)

B1  PMO^(cp)

B2  PMO^(cps)

B3  PMO^(cpr)

B4  PMO^(Shc)

B5  PMO^(morpholino) (m)

B6  PMO^(tri) (t)

B7  PMO^(hex) (h)

B8  PMO^(dodec)

B9  PMO^(dihex)

B10 PMO^(apn) (a)

B11 PMO^(pyr) (p)

B12 PMO^(pyr) (HCl Salt)

B13 PMO^(rba)

B14 PMO^(sba)

B15 PMO^(dimethylapn)

B16 PMO^(etpip)

B17 PMO^(iprpip)

B18 PMO^(pyrQMe)

B19 PMO^(cb)

B20 PMO^(ma)

B21 PMO^(bu)

B22 PMO^(bi)

B23 PMO^(pip)

B24 PMO^(odmb)

B25 PMO^(tfb)

B26 PMO^(ctfb)

B27 PMO^(ptfb)

B28 PMO^(dcd)

B29 PMO^(dmb)

B30 PMO^(hy)

B31 PMO^(6ce)

B32 PMO^(b)

B33 PMO^(q)

B34 PMO^(npp)

B35 PMO^(o)

B36 PMO^(4ce)

B37 PMO^(5ce)

B38 PMO^(f3p)

B39 PMO^(cyp)

B40 PMO^(mop)

B41 PMO^(pp)

B42 PMO^(dmepip)

B43 PMO^(NPpip)

B44 PMO^(bipip)

B45 PMO^(suc)

 46 PMO^(glutaric)

B47 PMO^(tet)

B48 PMO^(thiol) (SH)

B49 PMO^(pros)

B50 PMO^(pror)

B51 PMO^(tme)

B52 PMO^(ca)

B53 PMO^(dca)

B54 PMO^(guan) (g)

B55 PMO^(+phos)

B56 PMO^(apnphos)

In the sequences and discussion that follows, the above names for thelinkages are often used. For example, a base comprising a PMO^(apn)linkage is illustrated as ^(apn)B, where B is a base. Other linkages aredesignated similarly. In addition, abbreviated designations may be used,for example, the abbreviated designations in parentheses above may beused (e.g., ^(a)B, refers to ^(apn)B). Other readily identifiableabbreviations may also be used.

B. Oligomers with Modified Terminal Groups

As noted above, the present disclosure also provides an oligomercomprising modified terminal groups. Applicants have found thatmodification of the 3′ and/or 5′ end of the oligomer with variouschemical moieties provides beneficial therapeutic properties (e.g.,enhanced cell delivery, potency, and/or tissue distribution, etc.) tothe oligomers. In various embodiments, the modified terminal groupscomprise a hydrophobic moiety, while in other embodiments the modifiedterminal groups comprise a hydrophilic moiety. The modified terminalgroups may be present with or without the linkages described above. Forexample, in some embodiments, the oligomers comprise one or moremodified terminal group and linkages of type (A), for example linkageswherein X is —N(CH₃)₂. In other embodiments, the oligomers comprise oneor more modified terminal group and linkages of type (B), for examplelinkages wherein X is 4-aminopiperidin-1-yl (i.e., APN). In yet otherembodiments, the oligomers comprise one or more modified terminal groupand a mixture of linkages (A) and (B). For example, the oligomers maycomprise one or more modified terminal group (e.g., trityl or triphenylacetyl) and linkages wherein X is —N(CH₃)₂ and linkages wherein X is4-aminopiperidin-1-yl. Other combinations of modified terminal groupsand modified linkages also provide favorable therapeutic properties tothe oligomers.

In one embodiment, the oligomers comprising terminal modifications havethe following structure (XVII):

or a salt or isomer thereof, wherein X, W and Y are as defined above forany of linkages (A) and (B) and:

R¹⁷ is, at each occurrence, independently absent, hydrogen or C₁-C₆alkyl;

R¹⁸ and R¹⁹ are, at each occurrence, independently absent, hydrogen, acell-penetrating peptide, a natural or non-natural amino acid, C₂-C₃₀alkylcarbonyl, —C(═O)OR²¹ or R²⁰;

R²⁰ is, at each occurrence, independently guanidinyl, heterocyclyl,C₁-C₃₀ alkyl, C₃-C₈ cycloalkyl; C₆-C₃₀ aryl, C₇-C₃₀ aralkyl, C₃-C₃₀alkylcarbonyl, C₃-C₈ cycloalkylcarbonyl, C₃-C₈ cycloalkylalkylcarbonyl,C₇-C₃₀ arylcarbonyl, C₇-C₃₀ aralkylcarbonyl, C₂-C₃₀ alkyloxycarbonyl,C₃-C₈ cycloalkyloxycarbonyl, C₇-C₃₀ aryloxycarbonyl, C₈-C₃₀aralkyloxycarbonyl, or —P(═O)(R²²)₂;

B is a base-pairing moiety;

L¹ is an optional linker up to 18 atoms in length comprising bondsselected from alkyl, hydroxyl, alkoxy, alkylamino, amide, ester,disulfide, carbonyl, carbamate, phosphorodiamidate, phosphoroamidate,phosphorothioate, piperazine and phosphodiester; and

x is an integer of 0 or greater; and wherein at least one of R¹⁸ or R¹⁹is R²⁰; and

wherein at least one of R¹⁸ or R¹⁹ is R²⁰ and provided that both of R¹⁷and R¹⁸ are not absent.

The oligomers with modified terminal groups may comprise any number oflinkages of types (A) and (B). For example, the oligomers may compriseonly linkage type (A). For example, X in each linkage may be —N(CH₃)₂.Alternatively, the oligomers may only comprise linkage (B). In certainembodiments, the oligomers comprise a mixture of linkages (A) and (B),for example from 1 to 4 linkages of type (B) and the remainder of thelinkages being of type (A). Linkages in this regard include, but are notlimited to, linkages wherein X is aminopiperidinyl for type (B) anddimethyl amino for type (A).

In some embodiments, R¹⁷ is absent. In some embodiments, R¹⁷ ishydrogen. In some embodiments, R¹⁷ is C₁-C₆ alkyl. In some embodiments,R¹⁷ is methyl. In yet other embodiments, R¹⁷ is ethyl. In someembodiments, R¹⁷ is C₃ alkyl. In some other embodiments, R¹⁷ isisopropyl. In other embodiments, R¹⁷ is C₄ alkyl. In yet otherembodiments, R¹⁷ is C₅ alkyl. In some other embodiments, R¹⁷ is C₆alkyl.

In other embodiments, R¹⁸ is absent. In some embodiments, R¹⁸ ishydrogen. In some embodiments, R¹⁸ is a cell-penetrating peptide asdescribed in more detail below. In some embodiments, R¹⁸ is a natural ornon-natural amino acid, for example trimethylglycine. In someembodiments, R¹⁸ is R²⁰.

In other embodiments, R¹⁹ is absent. In some embodiments, R¹⁹ ishydrogen. In some embodiments, R¹⁹ is a cell-penetrating peptide asdescribed in more detail below. In some embodiments, R¹⁹ is a natural ornon-natural amino acid, for example trimethylglycine. In someembodiments, R¹⁹ is —C(═O)OR¹⁷, for example R¹⁹ may have the followingstructure:

In other embodiments R¹⁸ or R¹⁹ is C₂-C₃₀ alkylcarbonyl, for example—C(═O)(CH₂)_(n)CO₂H, where n is 1 to 6, for example 2. In otherexamples, R¹⁸ or R¹⁹ is acetyl.

In some embodiments, R²⁰ is, at each occurrence, independentlyguanidinyl, heterocyclyl, C₁-C₃₀ alkyl, C₃-C₈ cycloalkyl; C₆-C₃₀ aryl,C₇-C₃₀ aralkyl, C₃-C₃₀ alkylcarbonyl, C₃-C₈ cycloalkylcarbonyl, C₃-C₈cycloalkylalkylcarbonyl, C₆-C₃₀ arylcarbonyl, C₇-C₃₀ aralkylcarbonyl,C₂-C₃₀ alkyloxycarbonyl, C₃-C₈ cycloalkyloxycarbonyl, C₇-C₃₀aryloxycarbonyl, C₈-C₃₀ aralkyloxycarbonyl, —C(═O)OR²⁰, or —P(═O)(R²²)₂,wherein R²¹ is C₁-C₃₀ alkyl comprising one or more oxygen or hydroxylmoieties or combinations thereof and each R²² is C⁶-C¹² aryloxy.

In certain other embodiments, R¹⁹ is —C(═O)OR²¹ and R¹⁸ is hydrogen,guanidinyl, heterocyclyl, C₁-C₃₀ alkyl, C₃-C₈ cycloalkyl; C₆-C₃₀ aryl,C₃-C₃₀ alkylcarbonyl, C₃-C₈ cycloalkylcarbonyl, C₃-C₈cycloalkylalkylcarbonyl, C₇-C₃₀ arylcarbonyl, C₇-C₃₀ aralkylcarbonyl,C₂-C₃₀ alkyloxycarbonyl, C₃-C₈ cycloalkyloxycarbonyl, C₇-C₃₀aryloxycarbonyl, C₈-C₃₀ aralkyloxycarbonyl, or —P(═O)(R²²)₂, whereineach R²² is C⁶-C¹² aryloxy.

In other embodiments, R²⁰ is, at each occurrence, independentlyguanidinyl, heterocyclyl, C₁-C₃₀ alkyl, C₃-C₈ cycloalkyl; C₆-C₃₀ aryl,C₃-C₃₀ alkylcarbonyl, C₃-C₈ cycloalkylcarbonyl, C₃-C₈cycloalkylalkylcarbonyl, C₇-C₃₀ arylcarbonyl, C₇-C₃₀ aralkylcarbonyl,C₂-C₃₀ alkyloxycarbonyl, C₃-C₈ cycloalkyloxycarbonyl, C₇-C₃₀aryloxycarbonyl, C₈-C₃₀ aralkyloxycarbonyl, or —P(═O)(R²²)₂. While inother examples, R²⁰ is, at each occurrence, independently guanidinyl,heterocyclyl, C₁-C₃₀ alkyl, C₃-C₈ cycloalkyl; C₆-C₃₀ aryl, C₇-C₃₀aralkyl, C₃-C₈ cycloalkylcarbonyl, C₃-C₈ cycloalkylalkylcarbonyl, C₇-C₃₀arylcarbonyl, C₇-C₃₀ aralkylcarbonyl, C₂-C₃₀ alkyloxycarbonyl, C₃-C₈cycloalkyloxycarbonyl, C₇-C₃₀ aryloxycarbonyl, C₈-C₃₀aralkyloxycarbonyl, or —P(═O)(R²²)₂.

In some embodiments R²⁰ is guanidinyl, for example mono methylguanidynylor dimethylguanidinyl. In other embodiments, R²⁰ is heterocyclyl. Forexample, in some embodiments, R²⁰ is piperidin-4-yl. In someembodiments, the piperidin-4-yl is substituted with trityl or Bocgroups. In other embodiments, R²⁰ is C₃-C₈ cycloalkyl. In otherembodiments, R²⁰ is C₆-C₃₀ aryl.

In some embodiments, R²⁰ is C₇-C₃₀ arylcarbonyl. For example, In someembodiments, R²⁰ has the following structure (XVIII):

wherein R²³ is, at each occurrence, independently hydrogen, halo, C₁-C₃₀alkyl, C₁-C₃₀ alkoxy, C₁-C₃₀ alkyloxycarbonyl, C₇-C₃₀ aralkyl, aryl,heteroaryl, heterocyclyl or heterocyclalkyl, and wherein one R²³ mayjoin with another R²³ to form a heterocyclyl ring. In some embodiments,at least one R²³ is hydrogen, for example, in some embodiments, each R²³is hydrogen. In other embodiments, at least one R²³ is C₁-C₃₀ alkoxy,for example in some embodiments, each R²³ is methoxy. In otherembodiments, at least one R²³ is heteroaryl, for example in someembodiments, at least one R²³ has one of the following structures(XVIIIa) of (XVIIIb):

In still other embodiments, one R²³ joins with another R²³ to form aheterocyclyl ring. For example, in one embodiment, R²⁰ is5-carboxyfluorescein.

In other embodiments, R²⁰ is C₇-C₃₀ aralkylcarbonyl. For example, invarious embodiments, R²⁰ has one of the following structures (XIX), (XX)or (XXI):

wherein R²³ is, at each occurrence, independently hydrogen, halo, C₁-C₃₀alkyl, C₁-C₃₀ alkoxy, C₁-C₃₀ alkyloxycarbonyl, C₇-C₃₀ aralkyl, aryl,heteroaryl, heterocyclyl or heterocyclalkyl, wherein one R²³ may joinwith another R²³ to form a heterocyclyl ring, X is —OH or halo and m isan integer from 0 to 6. In some specific embodiments, m is 0. In otherembodiments, m is 1, while in other embodiments, m is 2. In otherembodiments, at least one R²³ is hydrogen, for example in someembodiments each R²³ is hydrogen. In some embodiments, X is hydrogen. Inother embodiments, X is —OH. In other embodiments, X is Cl. In otherembodiments, at least one R²³ is C₁-C₃₀ alkoxy, for example methoxy.

In still other embodiments, R²⁰ is C₇-C₃₀ aralkyl, for example trityl.In other embodiments, R²⁰ is methoxy trityl. In some embodiments, R²⁰has the following structure (XXII):

wherein R²³ is, at each occurrence, independently hydrogen, halo, C₁-C₃₀alkyl, C₁-C₃₀ alkoxy, C₁-C₃₀ alkyloxycarbonyl, C₇-C₃₀ aralkyl, aryl,heteroaryl, heterocyclyl or heterocyclalkyl, and wherein one R²³ mayjoin with another R²³ to form a heterocyclyl ring. For example, in someembodiments each R²³ is hydrogen. In other embodiments, at least one R²³is C₁-C₃₀ alkoxy, for example methoxy.

In yet other embodiments, R²⁰ is C₇-C₃₀ aralkyl and R²⁰ has thefollowing structure (XXIII):

In some embodiments, at least one R²³ is halo, for example chloro. Insome other embodiments, one R²³ is chloro in the para position.

In other embodiments, R²⁰ is C₁-C₃₀ alkyl. For example, In someembodiments, R²⁰ is a C₄-C₂₀ alkyl and optionally comprises one or moredouble bonds. For example, In some embodiments, R²⁰ is a C₄₋₁₀ alkylcomprising a triple bond, for example a terminal triple bond. In someembodiments, R²⁰ is hexyn-6-yl. In some embodiments, R²⁰ has one of thefollowing structures (XXIV), (XXV), (XXVI) or (XXVII):

In still other embodiments, R²⁰ is a C₃-C₃₀ alkylcarbonyl, for example aC₃-C₁₀ alkyl carbonyl. In some embodiments, R²⁰ is —C(═O)(CH₂)_(p)SH or—C(═O)(CH₂)_(p)SSHet, wherein p is an integer from 1 to 6 and Het is aheteroaryl. For example, p may be 1 or p may be 2. In other example Hetis pyridinyl, for example pyridin-2-yl. In other embodiments, the C₃-C₃₀alkylcarbonyl is substituted with a further oligomer, for example insome embodiments the oligomer comprises a C₃-C₃₀ alkyl carbonyl at the3′ position which links the oligomer to the 3′ position of anotheroligomer. Such terminal modifications are included within the scope ofthe present disclosure.

In other embodiments, R²⁰ is a C₃-C₃₀ alkyl carbonyl which is furthersubstituted with an arylphosphoryl moiety, for example triphenylphosphoryl. Examples of such R²⁰ groups include structure 33 in Table 2.

In other examples, R₂₀ is C₃-C₈ cycloalkylcarbonyl, for example C₅-C₇alkyl carbonyl. In these embodiments, R₂₀ has the following structure(XXVIII):

wherein R²³ is, at each occurrence, independently hydrogen, halo, C₁-C₃₀alkyl, C₁-C₃₀ alkoxy, C₁-C₃₀ alkyloxycarbonyl, C₇-C₃₀ aralkyl, aryl,heteroaryl, heterocyclyl or heterocyclalkyl, and wherein one R²³ mayjoin with another R²³ to form a heterocyclyl ring. In some embodiments,R²³ is heterocyclylalkyl, for example in some embodiments R²³ has thefollowing structure:

In some other embodiments, R²⁰ is C₃-C₈ cycloalkylalkylcarbonyl. Inother embodiments, R²⁰ is C₂-C₃₀ alkyloxycarbonyl. In other embodiments,R²⁰ is C₃-C₅ cycloalkyloxycarbonyl. In other embodiments, R²⁰ is C₇-C₃₀aryloxycarbonyl. In other embodiments, R²⁰ is C₈-C₃₀ aralkyloxycarbonyl.In other embodiments, R²⁰ is —P(═O)(R²²)₂, wherein each R²² is C⁶-C¹²aryloxy, for example in some embodiments R²⁰ has the following structure(C24):

In other embodiments, R²⁰ comprises one or more halo atoms. For example,in some embodiments R²⁰ comprises a perfluoro analogue of any of theabove R²⁰ moieties. In other embodiments, R²⁰ isp-trifluoromethylphenyl, trifluoromethyltrityl, perfluoropentyl orpentafluorophenyl.

In some embodiments the 3′ terminus comprises a modification and inother embodiments the 5′ terminus comprises a modification. In otherembodiments both the 3′ and 5′ termini comprise modifications.Accordingly, in some embodiments, R¹⁸ is absent and R¹⁹ is R²⁰. In otherembodiments, R¹⁹ is absent and R′8 is R²⁰. In yet other embodiments, R¹⁸and R¹⁹ are each R²⁰.

In some embodiments, the oligomer comprises a cell-penetrating peptidein addition to a 3′ or 5′ modification. Accordingly, in some embodimentsR¹⁹ is a cell-penetrating peptide and R¹⁸ is R²⁰. In other embodiments,R¹⁸ is a cell-penetrating peptide and R¹⁹ is R²⁰. In further embodimentsof the foregoing, the cell-penetrating peptide is an arginine-richpeptide.

In some embodiments, the linker L¹ which links the 5′ terminal group(i.e., R¹⁹) to the oligomer may be present or absent. The linkercomprises any number of functional groups and lengths provided thelinker retains its ability to link the 5′ terminal group to the oligomerand provided that the linker does not interfere with the oligomer'sability to bind to a target sequence in a sequence specific manner. Inone embodiment, L comprises phosphorodiamidate and piperazine bonds. Forexample, in some embodiments L has the following structure (XXIX):

wherein R²⁴ is absent, hydrogen or C₁-C₆ alkyl. In some embodiments, R²⁴is absent. In some embodiments, R²⁴ is hydrogen. In some embodiments,R²⁴ is C₁-C₆ alkyl. In some embodiments, R²⁴ is methyl. In otherembodiments, R²⁴ is ethyl. In yet other embodiments, R²⁴ is C₃ alkyl. Insome other embodiments, R²⁴ is isopropyl. In yet other embodiments, R²⁴is C₄ alkyl. In some embodiments, R²⁴ is C₅ alkyl. In yet otherembodiments, R²⁴ is C₆ alkyl.

In yet other embodiments, R²⁰ is C₃-C₃₀ alkylcarbonyl, and R²⁰ has thefollowing structure (XXX):

wherein R²⁵ is hydrogen or —SR²⁶, wherein R²⁶ is hydrogen, C₁-C₃₀ alkyl,heterocyclyl, aryl or heteroaryl, and q is an integer from 0 to 6.

In further embodiments of any of the above, R²³ is, at each occurrence,independently hydrogen, halo, C₁-C₃₀ alkyl, C₁-C₃₀ alkoxy, aryl,heteroaryl, heterocyclyl or heterocyclalkyl.

In some other embodiments, only the 3′ terminus of the oligomer isconjugated to one of the groups noted above. In some other embodiments,only the 5′ terminus of the oligomer is conjugated to one of the groupsnoted above. In other embodiments, both the 3′ and 5′ termini compriseone of the groups noted above. The terminal group may be selected fromany one of the groups noted above or any of the specific groupsillustrated in Table 2.

TABLE 2 Representative Terminal Groups No. Name Structure C1 Trimethoxybenzoyl

C2  9-fluorene-carboxyl

C3  4-carbazolylbenzoyl

C4  4-indazolylonebenzoyl

C5  Farnesyl

C6  Geranyl

C7  Prenyl

C8  Diphenylacetyl

C9  Chlorodiphenylacetyl

C10 Hydroxydiphenylacetyl

C11 Triphenylpropionyl

C12 Triphenylpropyl

C13 Triphenylacetyl

C14 Trityl (Tr)

C15 Methoxytrityl (MeOTr)

C16 Methylsuccinimidyl- cyclohexoyl

C17 Thioacetyl

C18 COCH₂CH₂SSPy

C19 Guanidinyl

C20 Trimethylglycine

C21 Lauroyl

C22 Triethyleneglycoloyl (EG3)

C23 Succinicacetyl

C24 Diphenylphosphoryl

C25 Piperidin-4-yl

C26 Tritylpiperidin-4-yl

C27 Boc-Piperidin-4-yl

C28 Hexyn-6-yl

C29 5-carboxyfluorescein

C30 Benzhydryl

C31 p-Chlorobenzhydryl

C32 Piperazinyl (pip)

C33 Triphenylphos

C34 Dimerized

1. Peptide Transporters

In some embodiments, the subject oligomer is conjugated to a peptidetransporter moiety, for example a cell-penetrating peptide transportmoiety, which is effective to enhance transport of the oligomer intocells. For example, in some embodiments the peptide transporter moietyis an arginine-rich peptide. In further embodiments, the transportmoiety is attached to either the 5′ or 3′ terminus of the oligomer, asshown, for example, in FIG. 1C. When such peptide is conjugated toeither termini, the opposite termini is then available for furtherconjugation to a modified terminal group as described herein.

In some embodiments of the foregoing, the peptide transport moietycomprises 6 to 16 subunits selected from X′ subunits, Y′ subunits, andZ′ subunits,

where

-   -   (a) each X′ subunit independently represents lysine, arginine or        an arginine analog, said analog being a cationic α-amino acid        comprising a side chain of the structure R³³N═C(NH₂)R³⁴, where        R³³ is H or R; R³⁴ is R³⁵, NH₂, NHR, or NR₃₄, where R³⁵ is lower        alkyl or lower alkenyl and may further include oxygen or        nitrogen; R³³ and R³⁴ may together form a ring; and the side        chain is linked to said amino acid via R³³ or R³⁴;    -   (b) each Y′ subunit independently represents a neutral amino        acid —C(O)—(CHR)_(n)—NH—, where n is 2 to 7 and each R is        independently H or methyl; and    -   (c) each Z′ subunit independently represents an α-amino acid        having a neutral aralkyl side chain;    -   wherein the peptide comprises a sequence represented by one of        (X′Y′X′)_(p), (X′Y′)_(m), and (X′Z′Z′)_(p), where p is 2 to 5        and m is 2 to 8.

In selected embodiments, for each X′, the side chain moiety is guanidyl,as in the amino acid subunit arginine (Arg). In further embodiments,each Y′ is —CO—(CH₂)_(n)—CHR—NH—, where n is 2 to 7 and R is H. Forexample, when n is 5 and R is H, Y′ is a 6-aminohexanoic acid subunit,abbreviated herein as Ahx; when n is 2 and R is H, Y′ is a β-alaninesubunit.

In certain embodiments, peptides of this type include those comprisingarginine dimers alternating with single Y′ subunits, where Y′ is Ahx.Examples include peptides having the formula (RY′R)_(p) or the formula(RRY′)_(p), where Y′ is Ahx. In one embodiment, Y′ is a 6-aminohexanoicacid subunit, R is arginine and p is 4.

In a further embodiment, each Z′ is phenylalanine, and m is 3 or 4.

In some embodiments, the conjugated peptide is linked to a terminus ofthe oligomer via a linker Ahx-B, where Ahx is a 6-aminohexanoic acidsubunit and B is a β-alanine subunit.

In selected embodiments, for each X′, the side chain moiety isindependently selected from the group consisting of guanidyl(HN═C(NH₂)NH—), amidinyl (HN═C(NH₂)C—), 2-aminodihydropyrimidyl,2-aminotetrahydropyrimidyl, 2-aminopyridinyl, and 2-aminopyrimidonyl,and it is preferably selected from guanidyl and amidinyl. In oneembodiment, the side chain moiety is guanidyl, as in the amino acidsubunit arginine (Arg).

In some embodiments, the Y′ subunits are either contiguous, in that noX′ subunits intervene between Y′ subunits, or interspersed singlybetween X′ subunits. However, in some embodiments the linking subunitmay be between Y′ subunits. In one embodiment, the Y′ subunits are at aterminus of the peptide transporter; in other embodiments, they areflanked by X′ subunits. In further embodiments, each Y′ is—CO—(CH₂)_(n)—CHR—NH—, where n is 2 to 7 and R is H. For example, when nis 5 and R is H, Y′ is a 6-aminohexanoic acid subunit, abbreviatedherein as Ahx. In selected embodiments of this group, each X′ comprisesa guanidyl side chain moiety, as in an arginine subunit. Exemplarypeptides of this type include those comprising arginine dimersalternating with single Y′ subunits, where Y′ is preferably Ahx.Examples include peptides having the formula (RY′R)₄ or the formula(RRY′)₄, where Y′ is preferably Ahx. In some embodiments, the nucleicacid analog is linked to a terminal Y′ subunit, preferably at theC-terminus, as shown, for example, in FIG. 1C. In other embodiments, thelinker is of the structure AhxB, where Ahx is a 6-aminohexanoic acidsubunit and B is a (3-alanine subunit.

The peptide transport moieties as described above have been shown togreatly enhance cell entry of attached oligomers, relative to uptake ofthe oligomer in the absence of the attached transport moiety, andrelative to uptake by an attached transport moiety lacking thehydrophobic subunits Y′. Such enhanced uptake may be evidenced by atleast a two-fold increase, or in other embodiments a four-fold increase,in the uptake of the compound into mammalian cells relative to uptake ofthe agent by an attached transport moiety lacking the hydrophobicsubunits Y′. In some embodiments, uptake is enhanced at least twentyfold or at least forty fold, relative to the unconjugated compound.

A further benefit of the peptide transport moiety is its expectedability to stabilize a duplex between an antisense oligomer and itstarget nucleic acid sequence. While not wishing to be bound by theory,this ability to stabilize a duplex may result from the electrostaticinteraction between the positively charged transport moiety and thenegatively charged nucleic acid. In some embodiments, the number ofcharged subunits in the transporter is less than 14, as noted above, orin other embodiments between 8 and 11, since too high a number ofcharged subunits may lead to a reduction in sequence specificity.

Exemplary arginine-rich cell-penetrating peptide transporters comprisinglinkers (B or AhxB) are given below in Table 3:

TABLE 3 Arginine-Rich Cell-Penetrating Peptide Transporters Name SEQ ID(Designation) Sequence NO.^(a) rTAT RRRQRRKKR 56 Tat RKKRRQRRR 57 R₉F₂RRRRRRRRRFF 58 R₅F₂R₄ RRRRRFFRRRR 59 R₄ RRRR 60 R₅ RRRRR 61 R₆ RRRRRR 62R₇ RRRRRRR 63 R₈ RRRRRRRR 64 R₉ RRRRRRRRR 65 (RAhxR)₄;RAhxRRAhxRRAhxRRAhxR 66 (P007) (RAhxR)₅; RAhxRRAhxRRAhxRRAhxRRAhxR 67(CP04057) (RAhxRRBR)₂; RAhxRRBRRAhxRRBR 68 (CP06062) (RAR)₄F₂RARRARRARRARFFC 69 (RGR)₄F₂ RGRRGRRGRRGRFFC 70

C. Properties of the Oligomers

As noted above, the present disclosure is directed to oligomercomprising various modifications which impart desirable properties(e.g., increased antisense activity) to the oligomers. In certainembodiments, the oligomer comprises a backbone comprising a sequence ofmorpholino ring structures joined by intersubunit linkages, theintersubunit linkages joining a 3′-end of one morpholino ring structureto a 5′-end of an adjacent morpholino ring structure, wherein eachmorpholino ring structure is bound to a base-pairing moiety, such thatthe oligomer can bind in a sequence-specific manner to a target nucleicacid. The morpholino ring structures may have the following structure(i):

wherein B is, at each occurrence, independently a base-pairing moiety.

Each morpholino ring structure supports a base pairing moiety (Pi), toform a sequence of base pairing moieties which is typically designed tohybridize to a selected antisense target in a cell or in a subject beingtreated. The base pairing moiety may be a purine or pyrimidine found innative DNA or RNA (A, G, C, T, or U) or an analog, such as hypoxanthine(the base component of the nucleoside inosine) or 5-methyl cytosine.Analog bases that confer improved binding affinity to the oligomer canalso be utilized. Exemplary analogs in this regard includeC5-propynyl-modified pyrimidines, 9-(aminoethoxy)phenoxazine (G-clamp)and the like.

As noted above, the oligomer may be modified, in accordance with anaspect of the invention, to include one or more (B) linkages, e.g. up toabout 1 per every 2-5 uncharged linkages, typically 3-5 per every 10uncharged linkages. Certain embodiments also include one or morelinkages of type (B). Optimal improvement in antisense activity is seenwhere up to about half of the backbone linkages are type (B). Some, butnot maximum enhancement is typically seen with a small number e.g.,10-20% of (B) linkages.

In one embodiment, the linkage types (A) and (B) are interspersed alongthe backbone. In some embodiments, the oligomer does not have a strictlyalternating pattern of (A) and (B) linkages along its entire length. Theoligomers may optionally comprise a 5′ and/or 3′ modification asdescribed above.

Also considered are oligomers having blocks of (A) linkages and blocksof (B) linkages; for example, a central block of (A) linkages may beflanked by blocks of (B) linkages, or vice versa. In one embodiment, theoligomer has approximately equal-length 5′, 3; and center regions, andthe percentage of (B) or (A) linkages in the center region is greaterthan about 50%, o greater than about 70%. Oligomers for use in antisenseapplications generally range in length from about 10 to about 40subunits, more preferably about 15 to 25 subunits. For example, anoligomer of the invention having 19-20 subunits, a useful length for anantisense oligomer, may ideally have two to seven, e.g. four to six, orthree to five, (B) linkages, and the remainder (A) linkages. An oligomerhaving 14-15 subunits may ideally have two to five, e.g. 3 or 4, (B)linkages and the remainder (A) linkages.

The morpholino subunits may also be linked by non-phosphorus-basedintersubunit linkages, as described further below, where at least onelinkage is linkage (B).

Other oligonucleotide analog linkages which are uncharged in theirunmodified state but which could also bear a pendant amine substituentcan also be used. For example, a 5′ nitrogen atom on a morpholino ringcould be employed in a sulfamide linkage (or a urea linkage, wherephosphorus is replaced with carbon or sulfur, respectively).

In some embodiments for antisense applications, the oligomer may be 100%complementary to the nucleic acid target sequence, or it may includemismatches, e.g., to accommodate variants, as long as a heteroduplexformed between the oligomer and nucleic acid target sequence issufficiently stable to withstand the action of cellular nucleases andother modes of degradation which may occur in vivo. Mismatches, ifpresent, are less destabilizing toward the end regions of the hybridduplex than in the middle. The number of mismatches allowed will dependon the length of the oligomer, the percentage of G:C base pairs in theduplex, and the position of the mismatch(es) in the duplex, according towell understood principles of duplex stability. Although such anantisense oligomer is not necessarily 100% complementary to the nucleicacid target sequence, it is effective to stably and specifically bind tothe target sequence, such that a biological activity of the nucleic acidtarget, e.g., expression of encoded protein(s), is modulated.

The stability of the duplex formed between an oligomer and the targetsequence is a function of the binding T_(m) and the susceptibility ofthe duplex to cellular enzymatic cleavage. The T_(m) of an antisensecompound with respect to complementary-sequence RNA may be measured byconventional methods, such as those described by Hames et al., NucleicAcid Hybridization, IRL Press, 1985, pp. 107-108 or as described inMiyada C. G. and Wallace R. B., 1987, Oligonucleotide hybridizationtechniques, Methods Enzymol. Vol. 154 pp. 94-107.

In some embodiments, each antisense oligomer has a binding T_(m), withrespect to a complementary-sequence RNA, of greater than bodytemperature or in other embodiments greater than 50° C. In otherembodiments T_(m)'s are in the range 60-80° C. or greater. According towell known principles, the T_(m) of an oligomer compound, with respectto a complementary-based RNA hybrid, can be increased by increasing theratio of C:G paired bases in the duplex, and/or by increasing the length(in base pairs) of the heteroduplex. At the same time, for purposes ofoptimizing cellular uptake, it may be advantageous to limit the size ofthe oligomer. For this reason, compounds that show high T_(m) (50° C. orgreater) at a length of 20 bases or less are generally preferred overthose requiring greater than 20 bases for high T_(m) values. For someapplications, longer oligomers, for example longer than 20 bases mayhave certain advantages. For example, in certain embodiments longeroligomers may find particular utility for use in exon skippin or splicemodulation.

The targeting sequence bases may be normal DNA bases or analoguesthereof, e.g., uracil and inosine that are capable of Watson-Crick basepairing to target-sequence RNA bases.

The oligomers may also incorporate guanine bases in place of adeninewhen the target nucleotide is a uracil residue. This is useful when thetarget sequence varies across different viral species and the variationat any given nucleotide residue is either cytosine or uracil. Byutilizing guanine in the targeting oligomer at the position ofvariability, the well-known ability of guanine to base pair with uracil(termed C/U:G base pairing) can be exploited. By incorporating guanineat these locations, a single oligomer can effectively target a widerrange of RNA target variability.

The compounds (e.g., oligomers, intersubunit linkages, terminal groups)may exist in different isomeric forms, for example structural isomers(e.g., tautomers). With regard to stereoisomers, the compounds may havechiral centers and may occur as racemates, enantiomerically enrichedmixtures, individual enantiomers, mixture or diastereomers or individualdiastereomers. All such isomeric forms are included within the presentinvention, including mixtures thereof. The compounds may also possessaxial chirality which may result in atropisomers. Furthermore, some ofthe crystalline forms of the compounds may exist as polymorphs, whichare included in the present invention. In addition, some of thecompounds may also form solvates with water or other organic solvents.Such solvates are similarly included within the scope of this invention.

The oligomers described herein may be used in methods of inhibitingproduction of a protein or replication of a virus. Accordingly, in oneembodiment a nucleic acid encoding such a protein is exposed to anoligomer as disclosed herein. In further embodiments of the foregoing,the antisense oligomer comprises either a 5′ or 3′ modified terminalgroup or combinations thereof, as disclosed herein, and the base pairingmoieties B form a sequence effective to hybridize to a portion of thenucleic acid at a location effective to inhibit production of theprotein. In one embodiment, the location is an ATG start codon region ofan mRNA, a splice site of a pre-mRNA, or a viral target sequence asdescribed below.

In one embodiment, the oligomer has a T_(m) with respect to binding tothe target sequence of greater than about 50° C., and it is taken up bymammalian cells or bacterial cells. In another embodiment, the oligomermay be conjugated to a transport moiety, for example an arginine-richpeptide, as described herein to facilitate such uptake. In anotherembodiment, the terminal modifications described herein can function asa transport moiety to facilitate uptake by mammalian and/or bacterialcells.

The preparation and properties of morpholino oligomers is described inmore detail below and in U.S. Pat. No. 5,185,444 and WO/2009/064471,each of which is hereby incorporated by reference in their entirety.

D. Formulation and Administration of the Oligomers

The present disclosure also provides for formulation and delivery of thedisclosed oligomer. Accordingly, in one embodiment the presentdisclosure is directed to a composition comprising an oligomer asdisclosed herein and a pharmaceutically acceptable vehicle.

Effective delivery of the antisense oligomer to the target nucleic acidis an important aspect of treatment. Routes of antisense oligomerdelivery include, but are not limited to, various systemic routes,including oral and parenteral routes, e.g., intravenous, subcutaneous,intraperitoneal, and intramuscular, as well as inhalation, transdermaland topical delivery. The appropriate route may be determined by one ofskill in the art, as appropriate to the condition of the subject undertreatment. For example, an appropriate route for delivery of anantisense oligomer in the treatment of a viral infection of the skin istopical delivery, while delivery of a antisense oligomer for thetreatment of a viral respiratory infection is by inhalation. Theoligomer may also be delivered directly to the site of viral infection,or to the bloodstream.

The antisense oligomer may be administered in any convenient vehiclewhich is physiologically and/or pharmaceutically acceptable. Such acomposition may include any of a variety of standard pharmaceuticallyacceptable carriers employed by those of ordinary skill in the art.Examples include, but are not limited to, saline, phosphate bufferedsaline (PBS), water, aqueous ethanol, emulsions, such as oil/wateremulsions or triglyceride emulsions, tablets and capsules. The choice ofsuitable physiologically acceptable carrier will vary dependent upon thechosen mode of administration.

The compounds (e.g., oligomers) of the present invention may generallybe utilized as the free acid or free base. Alternatively, the compoundsof this invention may be used in the form of acid or base additionsalts. Acid addition salts of the free amino compounds of the presentinvention may be prepared by methods well known in the art, and may beformed from organic and inorganic acids. Suitable organic acids includemaleic, fumaric, benzoic, ascorbic, succinic, methanesulfonic, acetic,trifluoroacetic, oxalic, propionic, tartaric, salicylic, citric,gluconic, lactic, mandelic, cinnamic, aspartic, stearic, palmitic,glycolic, glutamic, and benzenesulfonic acids. Suitable inorganic acidsinclude hydrochloric, hydrobromic, sulfuric, phosphoric, and nitricacids. Base addition salts included those salts that form with thecarboxylate anion and include salts formed with organic and inorganiccations such as those chosen from the alkali and alkaline earth metals(for example, lithium, sodium, potassium, magnesium, barium andcalcium), as well as the ammonium ion and substituted derivativesthereof (for example, dibenzylammonium, benzylammonium,2-hydroxyethylammonium, and the like). Thus, the term “pharmaceuticallyacceptable salt” of structure (I) is intended to encompass any and allacceptable salt forms.

In addition, prodrugs are also included within the context of thisinvention. Prodrugs are any covalently bonded carriers that release acompound of structure (I) in vivo when such prodrug is administered to apatient. Prodrugs are generally prepared by modifying functional groupsin a way such that the modification is cleaved, either by routinemanipulation or in vivo, yielding the parent compound. Prodrugs include,for example, compounds of this invention wherein hydroxy, amine orsulfhydryl groups are bonded to any group that, when administered to apatient, cleaves to form the hydroxy, amine or sulfhydryl groups. Thus,representative examples of prodrugs include (but are not limited to)acetate, formate and benzoate derivatives of alcohol and aminefunctional groups of the compounds of structure (I). Further, in thecase of a carboxylic acid (—COOH), esters may be employed, such asmethyl esters, ethyl esters, and the like.

In some instances, liposomes may be employed to facilitate uptake of theantisense oligonucleotide into cells. (See, e.g., Williams, S. A.,Leukemia 10(12):1980-1989, 1996; Lappalainen et al., Antiviral Res.23:119, 1994; Uhlmann et al., antisense oligonucleotides: a newtherapeutic principle, Chemical Reviews, Volume 90, No. 4, pages544-584, 1990; Gregoriadis, G., Chapter 14, Liposomes, Drug Carriers inBiology and Medicine, pp. 287-341, Academic Press, 1979). Hydrogels mayalso be used as vehicles for antisense oligomer administration, forexample, as described in WO 93/01286. Alternatively, theoligonucleotides may be administered in microspheres or microparticles.(See, e.g., Wu, G. Y. and Wu, C. H., J. Biol. Chem. 262:4429-4432,1987). Alternatively, the use of gas-filled microbubbles complexed withthe antisense oligomers can enhance delivery to target tissues, asdescribed in U.S. Pat. No. 6,245,747. Sustained release compositions mayalso be used. These may include semipermeable polymeric matrices in theform of shaped articles such as films or microcapsules.

In one embodiment, antisense inhibition is effective in treatinginfection of a host animal by a virus, by contacting a cell infectedwith the virus with an antisense agent effective to inhibit thereplication of the specific virus. The antisense agent is administeredto a mammalian subject, e.g., human or domestic animal, infected with agiven virus, in a suitable pharmaceutical carrier. It is contemplatedthat the antisense oligonucleotide arrests the growth of the RNA virusin the host. The RNA virus may be decreased in number or eliminated withlittle or no detrimental effect on the normal growth or development ofthe host.

In one aspect of the method, the subject is a human subject, e.g., apatient diagnosed as having a localized or systemic viral infection. Thecondition of a patient may also dictate prophylactic administration ofan antisense oligomer of the invention, e.g. in the case of a patientwho (1) is immunocompromised; (2) is a burn victim; (3) has anindwelling catheter; or (4) is about to undergo or has recentlyundergone surgery. In one preferred embodiment, the oligomer is aphosphorodiamidate morpholino oligomer, contained in a pharmaceuticallyacceptable carrier, and is delivered orally. In another preferredembodiment, the oligomer is a phosphorodiamidate morpholino oligomer,contained in a pharmaceutically acceptable carrier, and is deliveredintravenously (i.v.).

In another application of the method, the subject is a livestock animal,e.g., a chicken, turkey, pig, cow or goat, etc, and the treatment iseither prophylactic or therapeutic. The invention also includes alivestock and poultry food composition containing a food grainsupplemented with a subtherapeutic amount of an antiviral antisensecompound of the type described above. Also contemplated is, in a methodof feeding livestock and poultry with a food grain supplemented withsubtherapeutic levels of an antiviral, an improvement in which the foodgrain is supplemented with a subtherapeutic amount of an antiviraloligonucleotide composition as described above.

In one embodiment, the antisense compound is administered in an amountand manner effective to result in a peak blood concentration of at least200-400 nM antisense oligomer. Typically, one or more doses of antisenseoligomer are administered, generally at regular intervals, for a periodof about one to two weeks. Preferred doses for oral administration arefrom about 1-1000 mg oligomer per 70 kg. In some cases, doses of greaterthan 1000 mg oligomer/patient may be necessary. For i.v. administration,preferred doses are from about 0.5 mg to 1000 mg oligomer per 70 kg. Theantisense oligomer may be administered at regular intervals for a shorttime period, e.g., daily for two weeks or less. However, in some casesthe oligomer is administered intermittently over a longer period oftime. Administration may be followed by, or concurrent with,administration of an antibiotic or other therapeutic treatment. Thetreatment regimen may be adjusted (dose, frequency, route, etc.) asindicated, based on the results of immunoassays, other biochemical testsand physiological examination of the subject under treatment.

An effective in vivo treatment regimen using the antisenseoligonucleotides of the invention may vary according to the duration,dose, frequency and route of administration, as well as the condition ofthe subject under treatment (i.e., prophylactic administration versusadministration in response to localized or systemic infection).Accordingly, such in vivo therapy will often require monitoring by testsappropriate to the particular type of viral infection under treatment,and corresponding adjustments in the dose or treatment regimen, in orderto achieve an optimal therapeutic outcome. Treatment may be monitored,e.g., by general indicators of disease and/or infection, such ascomplete blood count (CBC), nucleic acid detection methods,immunodiagnostic tests, viral culture, or detection of heteroduplex.

The efficacy of an in vivo administered antiviral antisense oligomer ofthe invention in inhibiting or eliminating the growth of one or moretypes of RNA virus may be determined from biological samples (tissue,blood, urine etc.) taken from a subject prior to, during and subsequentto administration of the antisense oligomer. Assays of such samplesinclude (1) monitoring the presence or absence of heteroduplex formationwith target and non-target sequences, using procedures known to thoseskilled in the art, e.g., an electrophoretic gel mobility assay; (2)monitoring the amount of viral protein production, as determined bystandard techniques such as ELISA or Western blotting, or (3) measuringthe effect on viral titer, e.g. by the method of Spearman-Karber. (See,for example, Pari, G. S. et al., Antimicrob. Agents and Chemotherapy39(5):1157-1161, 1995; Anderson, K. P. et al., Antimicrob. Agents andChemotherapy 40(9):2004-2011, 1996, Cottral, G. E. (ed) in: Manual ofStandard Methods for Veterinary Microbiology, pp. 60-93, 1978).

In some embodiments, the oligomer is actively taken up by mammaliancells. In further embodiments, the oligomer may be conjugated to atransport moiety (e.g., transport peptide) as described herein tofacilitate such uptake.

E. Preparation of the Oligomers

The morpholino subunits, the modified intersubunit linkages andoligomers comprising the same can be prepared as described in theexamples and in U.S. Pat. Nos. 5,185,444 and 7,943,762 which are herebyincorporated by reference in their entirety. The morpholino subunits canbe prepared according to the following general Reaction Scheme I.

Referring to Reaction Scheme 1, wherein B represents a base pairingmoiety and PG represents a protecting group, the morpholino subunits maybe prepared from the corresponding ribinucleoside (1) as shown. Themorpholino subunit (2) may be optionally protected by reaction with asuitable protecting group precursor, for example trityl chloride. The 3′protecting group is generally removed during solid-state oligomersynthesis as described in more detail below. The base pairing poiety maybe suitable protected for sold phase oligomer synthesis. Suitableprotecting groups include benzoyl for adenine and cytosine, phenylacetylfor guanine, and pivaloyloxymethyl for hypoxanthine (I). Thepivaloyloxymethyl group can be introduced onto the N1 position of thehypoxanthine heterocyclic base. Although an unprotected hypoxanthinesubunit, may be employed, yields in activation reactions are farsuperior when the base is protected. Other suitable protecting groupsinclude those disclosed in co-pending U.S. application Ser. No.12/271,040, which is hereby incorporated by reference in its entirety.

Reaction of 3 with the activated phosphorous compound 4, results inmorpholino subunints having the desired linkage moiety (5). Compounds ofstructure 4 can be prepared using any number of methods known to thoseof skill in the art. For example, such compounds may be prepared byreaction of the corresponding amine and phosphorous oxychloride. In thisregard, the amine starting material can be prepared using any methodknown in the art, for example those methods described in the Examplesand in U.S. Pat. No. 7,943,762. Although the above scheme depictspreparation of linkages of type (B) (e.g., X is —NR⁸R⁹), linkages oftype (A) (e.g., X is dimethyl amine) can be prepared in an analogousmanner.

Compounds of structure 5 can be used in solid-phase automated oligomersynthesis for preparation of oligomers comprising the intersubunitlinkages. Such methods are well known in the art. Briefly, a compound ofstructure 5 may be modified at the 5′ end to contain a linker to a solidsupport. For example, compound 5 may be linked to a solid support by alinker comprising L¹ and/or R¹⁹. An exemplary method is demonstrated inFIGS. 3 and 4. In this manner, the oligo may comprise a 5′-terminalmodification after oligomer synthesis is complete and the oligomer iscleaved from the solid support. Once supported, the protecting group of5 (e.g., trityl) is removed and the free amine is reacted with anactivated phosphorous moiety of a second compound of structure 5. Thissequence is repeated until the desired length oligo is obtained. Theprotecting group in the terminal 5′ end may either be removed or left onif a 5′-modification is desired. The oligo can be removed from the solidsupport using any number of methods, or example treatment with a base tocleave the linkage to the solid support.

In one embodiment, the disclosure provides morpholino subunits forpreparation of the oligomers, as well as related methods. The morpholinosubunits have the following structure (XXXI)

Wherein W, X and Y are as defined for linkage (B) above, B is a basepairing moiety, Z is a linkage to a solid support or a suitable leavinggroup and PG is a protecting group, for example C₇-C₃₀ aralkyl. In someembodiments, PG is trityl, for example methoxytrityl. In otherembodiments, the linkage to the solid support comprises L² and/or R¹⁹ asdefined above. L² is an optional linker comprising bonds selected fromalkyl, hydroxyl, alkoxy, alkylamino, amide, ester, disulfide, carbonyl,carbamate, phosphorodiamidate, phosphoroamidate, phosphorothioate,piperazine and phosphodiester. The length of L² is not particularlylimited. In some embodiments, L² is less than 60 atoms in length, lessthan 50 atoms in length or less than 40 atoms length. In some otherembodiments, Z is halo, for example chloro.

In still another embodiment, the present disclosure provides a method ofpreparing any of the disclosed oligomers. The method comprises use of acompound of structure (XXXI) for preparation of the oligomer.

The preparation of modified morpholino subunits and morpholino oligomersare described in more detail in the Examples. The morpholino oligomerscontaining any number of modified linkages may be prepared using methodsdescribed herein, methods known in the art and/or described by referenceherein. Also described in the examples are global modifications of PMO+morpholino oligomers prepared as previously described (see e.g., PCTpublication WO2008036127).

F. Antisense Activity of the Oligomers

The present disclosure also provides a method of inhibiting productionof a protein, the method comprising exposing a nucleic acid encoding theprotein to an oligomer as disclosed herein. Accordingly, in oneembodiment a nucleic acid encoding such a protein is exposed to anantisense oligomer comprising at least one linkage of type (B), or inother embodiments 10% to 50% such modified linkages, as disclosedherein, where the base pairing moieties Pi form a sequence effective tohybridize to a portion of the nucleic acid at a location effective toinhibit production of the protein. The oligomer may target, for example,an ATG start codon region of an mRNA, a splice site of a pre-mRNA, or aviral target sequence as described below. In another embodiment, themethod comprises exposing a nucleic acid encoding such a protein to anantisense oligomer comprising at least one terminal modification (e.g.,at least one R²⁰ moiety).

In another embodiment, the disclosure provides a method of enhancingantisense activity of an oligomer having a sequence of morpholinosubunits, joined by intersubunit linkages, supporting base-pairingmoieties, the method comprises modifying an oligomer as described hereinto contain at least one of the modified terminal groups, at least oneintersubunit linkage of type (B) or combinations thereof.

In some embodiments, enhancement of antisense activity may be evidencedby:

(i) a decrease in expression of an encoded protein, relative to thatprovided by a corresponding unmodified oligomer, when binding of theantisense oligomer to its target sequence is effective to block atranslation start codon for the encoded protein, or

(ii) an increase in expression of an encoded protein, relative to thatprovided by a corresponding unmodified oligomer, when binding of theantisense oligomer to its target sequence is effective to block anaberrant splice site in a pre-mRNA which encodes said protein whencorrectly spliced. Assays suitable for measurement of these effects aredescribed further below. In one embodiment, modification provides thisactivity in a cell-free translation assay, a splice correctiontranslation assay in cell culture, or a splice correction gain offunction animal model system as described herein. In one embodiment,activity is enhanced by a factor of at least two, at least five or atleast ten.

Described below are various exemplary applications of the oligomers ofthe invention including antiviral applications, treatment ofneuromuscular diseases, bacterial infections, inflammation andpolycystic kidney disease. This description is not meant to limit theinvention in any way but serves to exemplify the range of human andanimal disease conditions that can be addressed using oligomerscomprising the modified intersubunit linkages described herein.

G. In Vitro Activity in Cell Free Assays

The oligomers with partially modified linkages, such as PMO^(apn) (b10)and PMO^(suc) (b45), have higher affinity for DNA and RNA than do thecorresponding neutral compounds, demonstrated by enhanced antisenseactivity in vitro and in vivo. The oligomers of the invention were shownto provide superior antisense activity compared to fully unmodifiedoligomers when directed to a variety of different targets. In a firstseries of experiments, various unmodified, modified andpeptide-conjugated PPMO targeting exon 23 of the MDX mouse dystrophingene were prepared, as described in Materials and Methods and Example27. The sequences are shown as in Example 27, with the previouslydescribed (1-piperazino) phosphinylideneoxy linkage (as shown in FIG.1B) at each position indicated with a “+” for SEQ ID NOs: 2-5; the4-aminopiperidinyl linkage (structure (b10); FIG. 2) indicated with an“^(a)” for SEQ ID NO: 5 or; the 4-succinamidopiperazinyl linkage(structure (b45); FIG. 2) indicated with an “^(s)”. As described inExample 27, PMO oligomers containing an exemplary linkage (e.g.,PMO^(apn)) of the invention were more active compared to previouslydescribed PMO+ compounds.

1. Targeting Stem-Loop Secondary Structure of ssRNA Viruses

One class of an exemplary antisense antiviral compound is a morpholinooligomer as described herein, for example and oligomer comprising atleast one linkage of type (B) and/or at least one terminal modification(e.g., at least one R²⁰) or combinations thereof, having a sequence of12-40 subunits and a targeting sequence that is complementary to aregion associated with stem-loop secondary structure within the5′-terminal end 40 bases of the positive-sense RNA strand of thetargeted virus. (See, e.g., PCT Pubn. No. WO/2006/033933 or U.S. Appn.Pubn. Nos. 20060269911 and 20050096291, which are incorporated herein byreference.)

The method comprises first identifying as a viral target sequence, aregion within the 5′-terminal 40 bases of the positive strand of theinfecting virus whose sequence is capable of forming internal stem-loopsecondary structure. There is then constructed, by stepwise solid-phasesynthesis, an oligomer comprising at least one linkage of type (B)and/or at least one terminal modification (e.g., at least one R²⁰) orcombinations thereof, and in other embodiments containing 20% to 50%such modified linkages, and having a targeting sequence of at least 12subunits that is complementary to the virus-genome region capable offorming internal duplex structure, where the oligomer is able to formwith the viral target sequence, a heteroduplex structure composed of thepositive sense strand of the virus and the oligonucleotide compound, andcharacterized by a Tm of dissociation of at least 45° C. and disruptionof such stem-loop structure.

The target sequence may be identified by analyzing the 5′-terminalsequences, e.g., the 5′-terminal 40 bases, by a computer program capableof performing secondary structure predictions based on a search for theminimal free energy state of the input RNA sequence.

In a related aspect, the oligomers can be used in methods of inhibitingin a mammalian host cell, replication of an infecting RNA virus having asingle-stranded, positive-sense genome and selected from one of theFlaviviridae, Picornoviridae, Caliciviridae, Togaviridae, Arteriviridae,Coronaviridae, Astroviridae or Hepeviridae families. The method includesadministering to the infected host cells, a virus-inhibitory amount ofan oligomer as described herein, having a targeting sequence of at least12 subunits that is complementary to a region within the 5′-terminal 40bases of the positive-strand viral genome that is capable of forminginternal stem-loop secondary structure. The compound is effective, whenadministered to the host cells, to form a heteroduplex structure (i)composed of the positive sense strand of the virus and theoligonucleotide compound, and (ii) characterized by a Tm of dissociationof at least 45° C. and disruption of such stem-loop secondary structure.The compound may be administered to a mammalian subject infected withthe virus, or at risk of infection with the virus.

Exemplary targeting sequences that target the terminal stem loopstructures of the dengue and Japanese encephalitis viruses are listedbelow as SEQ ID NOs: 1 and 2, respectively.

Additional exemplary targeting sequences that target the terminal stemloop structures of ssRNA viruses can also be found in U.S. applicationSer. No. 11/801,885 and PCT publication WO/2008/036127 which areincorporated herein by reference.

2. Targeting the First Open Reading Frame of ssRNA Viruses

A second class of exemplary antisense antiviral compounds for use ininhibition of growth of viruses of the picornavirus, calicivirus,togavirus, coronavirus, and flavivirus families having asingle-stranded, positive sense genome of less than 12 kb and a firstopen reading frame that encodes a polyprotein containing multiplefunctional proteins. In particular embodiments, the virus is an RNAvirus from the coronavirus family or a West Nile, Yellow Fever or Denguevirus from the flavivirus family. The inhibiting compounds compriseantisense oligomers described herein, for example oligomers comprisingat least one linkage of type (B) and/or at least one terminalmodification (e.g., at least one R²⁰) or combinations thereof, having atargeting base sequence that is substantially complementary to a viraltarget sequence which spans the AUG start site of the first open readingframe of the viral genome. In one embodiment of the method, the oligomeris administered to a mammalian subject infected with the virus. See,e.g., PCT Pubn. No. WO/2005/007805 and US Appn. Pubn. No. 2003224353,which are incorporated herein by reference.

The preferred target sequence is a region that spans the AUG start siteof the first open reading frame (ORF1) of the viral genome. The firstORF generally encodes a polyprotein containing non-structural proteinssuch as polymerases, helicases and proteases. By “spans the AUG startsite” is meant that the target sequence includes at least three bases onone side of the AUG start site and at least two bases on the other (atotal of at least 8 bases). Preferably, it includes at least four baseson each side of the start site (a total of at least 11 bases).

More generally, preferred target sites include targets that areconserved between a variety of viral isolates. Other favored sitesinclude the IRES (internal ribosome entry site), transactivation proteinbinding sites, and sites of initiation of replication. Complex and largeviral genomes, which may provide multiple redundant genes, may beefficiently targeted by targeting host cellular genes coding for viralentry and host response to viral presence.

A variety of viral-genome sequences are available from well knownsources, such as the NCBI Genbank databases. The AUG start site of ORF1may also be identified in the gene database or reference relied upon, orit may be found by scanning the sequence for an AUG codon in the regionof the expected ORF1 start site.

The general genomic organization of each of the four virus families isgiven below, followed by exemplary target sequences obtained forselected members (genera, species or strains) within each family.

3. Targeting Influenza Virus

A third class of exemplary antisense antiviral compounds are used ininhibition of growth of viruses of the Orthomyxoviridae family and inthe treatment of a viral infection. In one embodiment, the host cell iscontacted with an oligomer as described herein, for example an oligomercomprising at least one linkage of type (B) and/or at least one terminalmodification (e.g., at least one R²⁰) or combinations thereof, or inother embodiments comprising 20% to 50% such modified linkages, andcomprising a base sequence effective to hybridize to a target regionselected from the following: 1) the 5′ or 3′ terminal 25 bases of thenegative sense viral RNA segments; 2) the terminal 25 bases of the 5′ or3′ terminus of the positive sense cRNA; 3) 45 bases surrounding the AUGstart codons of influenza viral mRNAs and; 4) 50 bases surrounding thesplice donor or acceptor sites of influenza mRNAs subject to alternativesplicing. (See, e.g., PCT Pubn. No. WO/2006/047683; U.S. Appn. Pubn. No.20070004661; and PCT Appn. Num. 2010/056613 and U.S. application Ser.No. 12/945,081, which are incorporated herein by reference.)

Experiments in support of the invention and designed to target the M1/M2segment of influenza A virus (H1N1 strain PR8) using PMO with modifiedlinkages of the invention were performed using oligomers based on SEQ IDNO:3, listed below in Table 4 and described in Example 29.

TABLE 4 Influenza targeting sequences that  incorporate modified intersubunit linkages or terminal groups NG-10-0038PMOhex CGG T^(h) TA GAA GAC ^(h) TCA TC^(h) T TT NG-10-0039 PMOhexCGG T^(h) TA GAA GAC ^(h) TCA ^(h) TCT ^(h) TT NG-10-0096 PMOapnCGG T^(a) TA GAA GAC ^(a) TCA TC^(a) T TT NG-10-0097 PMOapn CGG ^(a) T^(a) TA GAA GAC ^(a) TCA ^(a) TC^(a) T TT NG-10-0099 PMOpyr CGG ^(p) T^(p) TA GAA GAC ^(p) TCA ^(p) TC^(p) T TT NG-10-0107 PMOthiol CGG T^(SH)TA GAA GAC ^(SH) TCA TC^(SH) T TT NG-10-0108 PMOsucc CGG T^(s)TA GAA GAC ^(s) TCA TC^(s) T TT NG-10-0111 PMOguan CGG T^(g) TA GAA GAC^(g) TCA TC^(g) T TT NG-10-0141 PMOpyr CGG T^(p) TA GAA GAC ^(p)TCA TC^(p) T TT NG-10-0142 PMOpyr CGG T^(p) TA GAA GAC ^(p) TCA ^(p)TC^(p) T TT NG-10-0158 PMOglutaric CGG T^(glu) TA GAA GAC ^(glu)TCA TC^(glu) T TT NG-10-0159 PMOcyclo-glut CGG T^(cpglu) TA GAA GAC^(cpglu) TCA TC^(cpglu) T TT NG-10-0160 PMOcholic acid CGG T^(ca)TA GAA GAC  ^(ca) TCA TC^(ca) T TT NG-10-0161 PMOdeoxyCA CGG T^(dca)TA GAA GAC ^(dca) TCA TC^(dca) T TT NG-10-0180 PMOapn TT^(a)T CGA CA^(a) T CGG T^(a) TA GAA GAC ^(a) TCA T NG-10-0174 PMOm CGG T^(m)TA GAA GAC ^(m) TCA TC^(m) T TT NG-10-0222 PMO MeT CGG T^(Me) TA GAA GAC+TCA TC+T TT NG-10-0223 PMO FarnT CGG T^(Farn) TA GAA GAC +TCA TC+T TTNG-10-0538 PMOapn-trityl CGG T^(a) TA GAA GAC ^(a) TCA TC^(a) T TTNG-10-0539 PMOapn-trityl CGG T^(p) TA GAA GAC ^(p) TCA TC^(p) T TTNG-10-0015 PMO CGG TTA GAA GAC TCA TCT TT NG-11-0170 PMOplusCGG +TTA GAA GAC +TCA TC+T TT NG-11-0145 PMOplus- CGG T+TA GAA GACbenzhydryl +TCA TC+T TT** NG-11-0148 PMOisopropylPip CGG TiprpipTA GAAGAC iprpipTCA  TCiprpipT TT NG-11-0173 PMOpyr CGG pTTA GAA GACpTCA TCpT TT NG-11-0291 Trimethyl Gly CGG T*+TA GAA GAC *+TCA TC*+T TT**3′-benzhydryl; *+ linkages are trimethyl glycine acylated at thePMOplus linkages; PMOm represents T bases with a methyl group on the3-nitrogen position.

The compounds are particularly useful in the treatment of influenzavirus infection in a mammal. The oligomer may be administered to amammalian subject infected with the influenza virus, or at risk ofinfection with the influenza virus.

4. Targeting Viruses of the Picornaviridae Family

A fourth class of exemplary antisense antiviral compounds are used ininhibition of growth of viruses of the Picornaviridae family and in thetreatment of a viral infection. The compounds are particularly useful inthe treatment of Enterovirus and/or Rhinovirus infection in a mammal. Inthis embodiment, the antisense antiviral compounds comprise morpholinooligomers, for example morpholino oligomers comprising at least onelinkage of type (B) and/or at least one terminal modification (e.g., atleast one R²⁰) or combinations thereof, and having a sequence of 12-40subunits, including at least 12 subunits having a targeting sequencethat is complementary to a region associated with viral RNA sequenceswithin one of two 32 conserved nucleotide regions of the viral 5′untranslated region. (See, e.g., PCT Pubn. Nos. WO/2007/030576 andWO/2007/030691 or copending and co-owned U.S. application Ser. Nos.11/518,058 and 11/517,757, which are incorporated herein by reference.)An exemplary targeting sequence is listed below as SEQ NO: 6.

5. Targeting Viruses of the Flavivirus Family

A fifth class of exemplary antisense antiviral compounds are used ininhibition of replication of a flavivirus in animal cells. An exemplaryantisense oligomer of this class is a morpholino oligomer comprising atleast one linkage of type (B) and/or at least one terminal modification(e.g., at least one R²⁰) or combinations thereof, between 8-40nucleotide bases in length and having a sequence of at least 8 basescomplementary to a region of the virus' positive strand RNA genome thatincludes at least a portion of the 5′-cyclization sequence (5′-CS) or3′-CS sequences of the positive strand flaviviral RNA. A highlypreferred target is the 3′-CS and an exemplary targeting sequence fordengue virus is listed below as SEQ ID NO: 7. (See, e.g., PCT Pubn. No.(WO/2005/030800) or copending and co-owned U.S. application Ser. No.10/913,996, which are incorporated herein by reference.)

6. Targeting Viruses of the Nidovirus Family

A sixth class of exemplary antisense antiviral compounds are used ininhibition of replication of a nidovirus in virus-infected animal cells.An exemplary antisense oligomer of this class is a morpholino oligomercomprising at least one linkage of type (B) and/or at least one terminalmodification (e.g., at least one R²⁰) or combinations thereof, asdescribed in the present disclosure, and containing between 8-25nucleotide bases, and has a sequence capable of disrupting base pairingbetween the transcriptional regulatory sequences (TRS) in the 5′ leaderregion of the positive-strand viral genome and negative-strand 3′subgenomic region (See, e.g., PCT Pubn. No. WO/2005/065268 or U.S. Appn.Pubn. No. 20070037763, which are incorporated herein by reference.)

7. Targeting of Filoviruses

In another embodiment, one or more oligomers as described herein can beused in a method of in inhibiting replication within a host cell of anEbola virus or Marburg virus, by contacting the cell with an oligomer asdescribed herein, for example and oligomer comprising at least onelinkage of type (B) and/or at least one terminal modification (e.g., atleast one R²⁰) or combinations thereof, or in other embodiments 20% to50% such modified linkages, and having a targeting base sequence that iscomplementary to a target sequence composed of at least 12 contiguousbases within an AUG start-site region of a positive-strand mRNA, asdescribed further below.

The filovirus viral genome is approximately 19,000 bases ofsingle-stranded RNA that is unsegmented and in the antisenseorientation. The genome encodes 7 proteins from monocistronic mRNAscomplementary to the vRNA.

Target sequences are positive-strand (sense) RNA sequences that span orare just downstream (within 25 bases) or upstream (within 100 bases) ofthe AUG start codon of selected Ebola virus proteins or the 3′ terminal30 bases of the minus-strand viral RNA. Preferred protein targets arethe viral polymerase subunits VP35 and VP24, although L, nucleoproteinsNP and VP30, are also contemplated. Among these early proteins arefavored, e.g., VP35 is favored over the later expressed L polymerase.

In another embodiment, one or more oligomers as described herein can beused in a method of in inhibiting replication within a host cell of anEbola virus or Marburg virus, by contacting the cell with an oligomer asdescribed herein, comprising at least one modified intersubunit linkage,or in other embodiments 20% to 50% such modified linkages, and having atargeting base sequence that is complementary to a target sequencecomposed of at least 12 contiguous bases within an AUG start-site regionof a positive-strand mRNA of the Filovirus mRNA sequences. (See, e.g.,PCT Pubn. No. WO/2006/050414 or U.S. Pat. Nos. 7,524,829 and 7,507,196,and continuation applications with U.S. application Ser. Nos.12/402,455; 12/402,461; 12/402,464; and 12/853,180 which areincorporated herein by reference.)

8. Targeting of Arenaviruses

In another embodiment, an oligomer as described herein can be used in amethod for inhibiting viral infection in mammalian cells by a species inthe Arenaviridae family. In one aspect, the oligomers can be used intreating a mammalian subject infected with the virus. (See, e.g., PCTPubn. No. WO/2007/103529 or U.S. Pat. No. 7,582,615, which areincorporated herein by reference.)

Table 5 is an exemplary list of targeted viruses targeted by oligomersof the invention as organized by their Old World or New World Arenavirusclassification.

TABLE 5 Targeted Arenaviruses Family Genus Virus Arenaviridae ArenavirusOld World Arenaviruses Lassa virus (LASV) Lymphocytic choriomeningitisvirus (LCMV) Mopeia virus (MOPV) New World Arenaviruses Guanarito virus(GTOV) Junín virus (JUNV) Machupo virus (MACV) Pichinide virus (PICV)Pirital virus (PIRV) Sabiá virus (SABV) Tacaribe virus (TCRV) WhitewaterArroyo virus (WWAV)

The genome of Arenaviruses consists of two single-stranded RNA segmentsdesignated S (small) and L (large). In virions, the molar ratio of S- toL-segment RNAs is roughly 2:1. The complete S-segment RNA sequence hasbeen determined for several arenaviruses and ranges from 3,366 to 3,535nucleotides. The complete L-segment RNA sequence has also beendetermined for several arenaviruses and ranges from 7,102 to 7,279nucleotides. The 3′ terminal sequences of the S and L RNA segments areidentical at 17 of the last 19 nucleotides. These terminal sequences areconserved among all known arenaviruses. The 5′-terminal 19 or 20nucleotides at the beginning of each genomic RNA are imperfectlycomplementary with each corresponding 3′ end. Because of thiscomplementarity, the 3′ and 5′ termini are thought to base-pair and formpanhandle structures.

Replication of the infecting virion or viral RNA (vRNA) to form anantigenomic, viral-complementary RNA (vcRNA) strand occurs in theinfected cell. Both the vRNA and vcRNA encode complementary mRNAs;accordingly, Arenaviruses are classified as ambisense RNA viruses,rather than negative- or positive-sense RNA viruses. The ambisenseorientation of viral genes are on both the L- and S-segments. The NP andpolymerase genes reside at the 3′ end of the S and L vRNA segments,respectively, and are encoded in the conventional negative sense (i.e.,they are expressed through transcription of vRNA or genome-complementarymRNAs). The genes located at the 5′ end of the S and L vRNA segments,GPC and Z, respectively, are encoded in mRNA sense but there is noevidence that they are translated directly from genomic vRNA. Thesegenes are expressed instead through transcription of genomic-sense mRNAsfrom antigenomes (i.e., the vcRNA), full-length complementary copies ofgenomic vRNAs that function as replicative intermediates.

An exemplary targeting sequence for the arenavirus family of viruses islisted below as SEQ ID NO: 8.

9. Targeting of Respiratory Syncytial Virus

Respiratory syncytial virus (RSV) is the single most importantrespiratory pathogen in young children. RSV-caused lower respiratoryconditions, such as bronchiolitis and pneumonia, often requirehospitalization in children less than one-year-old. Children withcardiopulmonary diseases and those born prematurely are especially proneto experience severe disorders from this infection. RSV infection isalso an important illness in elderly and high-risk adults, and it is thesecond-most commonly identified cause of viral pneumonia in olderpersons (Falsey, Hennessey et al. 2005). The World Health Organizationestimates that RSV is responsible for 64 million clinical infections and160 thousand deaths annually worldwide. No vaccines are currentlyavailable for the prevention of RSV infection. Although many majoradvances in our understanding of RSV biology, epidemiology,pathophysiology, and host-immune-response have occurred over the pastfew decades, there continues to be considerable controversy regardingthe optimum management of infants and children with RSV infection.Ribavirin is the only licensed antiviral drug for treating RSVinfection, but its use is limited to high-risk or severely-ill infants.The utility of Ribavirin has been limited by its cost, variableefficacy, and tendency to generate resistant viruses (Marquardt 1995;Prince 2001). The current need for additional effective anti-RSV agentsis well-acknowledged.

It is known that peptide conjugated PMO (PPMO) can be effective ininhibiting RSV both in tissue culture and in an in vivo animal modelsystem (Lai, Stein et al. 2008). Two antisense PPMOs, designed to targetthe sequence that includes the 5′-terminal region and translationstart-site region of RSV L mRNA, were tested for anti-RSV activity incultures of two human airway cell lines. One of them, (RSV-AUG-2; SEQ IDNO 10), reduced viral titers by >2.0 log₁₀. Intranasal (i.n.) treatmentof BALB/c mice with RSV-AUG-2 PPMO before the RSV inoculation produced areduction in viral titer of 1.2 log₁₀ in lung tissue at day 5postinfection (p.i.), and attenuated pulmonary inflammation at day 7postinfection. These data showed that RSV-AUG-2 provided potent anti-RSVactivity worthy of further investigation as a candidate for potentialtherapeutic application (Lai, Stein et al. 2008). Despite the successwith RSV-AUG-2 PPMO as described above, it is desirable to avoidincorporating peptide conjugation in an antisense anti-RSV therapeuticdue to toxicity concerns and cost of goods considerations. Therefore, inanother embodiment of the present invention, one or more oligomers asdescribed herein can be used in a method of inhibiting replicationwithin a host cell of RSV, by contacting the cell with an oligomer asdescribed herein, for example an oligomer comprising at least onelinkage of type (B) and/or at least one terminal modification (e.g., atleast one R²⁰) or combinations thereof, or in other embodiments 10% to50% such modified linkages, and having a targeting base sequence that iscomplementary to a target sequence composed of at least 12 contiguousbases within an AUG start-site region of anmRNA from RSV, as describedfurther below.

The L gene of RSV codes for a critical component of the viral RNAdependent RNA polymerase complex. Antisense PPMO designed against thesequence spanning the AUG translation start-site codon of the RSV L genemRNA in the form of RSV-AUG-2 PPMO is complementary to sequence from the‘gene-start’ sequence (GS) present at the 5′ terminus of the L mRNA to13 nt into the coding sequence. A preferred L gene targeting sequence istherefore complementary to any 12 contiguous bases from the 5′ end ofthe L gene mRNA extending 40 bases in the 3′ direction or 22 bases intothe L gene coding sequence as shown below in Table 3 as SEQ ID NO: 9.Exemplary RSV L gene targeting sequences are listed below in Table 3 asSEQ ID NOs: 10-14. Any of the intersubunit modifications of theinvention described herein can be incorporated in the oligomers toprovide increased antisense activity, improved intracellular deliveryand/or tissue specificity for improved therapeutic activity. Exemplaryoligomers containing intersubunit linkages of the invention are listedbelow in Table 6.

TABLE 6 RSV target and targeting sequences SEQ Name Sequence (5′ to 3′)ID NO L target GGGACAAAATGGATCCCATTA  9 TTAATGGAAATTCTGCTAA RSV-AUG-2TAATGGGATCCATTTTGTCCC 10 RSV-AUG3 AATAATGGGATCCATTTTGTC 11 CC RSV-AUG4CATTAATAATGGGATCCATTT 12 TGTCCC RSV-AUG5 GAATTTCCATTAATAATGGGA 13TCCATTTTG RSV-AUG6 CAGAATTTCCATTAATAATGG 14 GATCCATT RSV-AUG3apn*AATAA^(apn)TGGGA^(apn)TCCA^(apn)T 11 T^(apn)TTG^(apn)TCCC RSV-AUG3guanAATAA^(guan)TGGGA^(guan)TCCA 11 ^(guan)TT^(guan)TTG^(guan)TCCC

10. Neuromuscular Diseases

In another embodiment, a therapeutic oligomer is provided for use intreating a disease condition associated with a neuromuscular disease ina mammalian subject. Exemplary intersubunit oligomer modifications shownto enhance transport into muscle tissue include those havingintersubunit linkages of structure b6, b10, b51 and b54. Antisenseoligomers that incorporate such linkages into the M23D antisenseoligomer (SEQ ID NO: 16) are tested for activity in the MDX mouse modelfor Duchene Muscular Dystrophy (DMD) as described in the Examples.Exemplary oligomers that incorporate the linkages used in someembodiments are listed below in Table 7. In some embodiments, thetherapeutic compound may be selected from the group consisting of:

(a) an antisense oligomer targeted against human myostatin, having abase sequence complementary to at least 12 contiguous bases in a targetregion of the human myostatin mRNA identified by SEQ ID NO: 18, fortreating a muscle wasting condition, as described previously (See, e.g.,U.S. patent application Ser. No. 12/493,140, which is incorporatedherein by reference; and PCT publication WO2006/086667). Exemplarymurine targeting sequences are listed as SEQ ID NOs: 19-20.

(b) an antisense oligomer capable of producing exon skipping in the DMDprotein (dystrophin), such as a PMO having a sequence selected from SEQID NOs: 22 to 35, to restore partial activity of the dystrophin protein,for treating DMD, as described previously (See, e.g., PCT Pubn. Nos.WO/2010/048586 and WO/2006/000057 or U.S. Patent Publication No. U.S.Ser. No. 09/061,960 all of which are incorporated herein by reference).

Several other neuromuscular diseases can be treated using the modifiedlinkages and terminal groups of the present invention. Exemplarycompounds for treating spinal muscle atrophy (SMA) and myotonicdystrophy (DM) are discussed below.

SMA is an autosomal recessive disease caused by chronic loss ofalpha-motor neurons in the spinal cord and can affect both children andadults. Reduced expression of survival motor neuron (SMN) is responsiblefor the disease (Hua, Sahashi et al. 2010). Mutations that cause SMA arelocated in the SMN1 gene but a paralogous gene, SMN2, can allowviability by compensating for loss of SMN1 if expressed from analternative splice form lacking exon 7 (delta7 SMN2). Antisensecompounds targeted to inton 6, exon 7 and intron 7 have all been shownto induce exon 7 inclusion to varying degrees. Antisense compoundstargeted to intron 7 are preferred (see e.g., PCT Publication Nos.WO/2010/148249, WO/2010/120820, WO/2007/002390 and U.S. Pat. No.7,838,657). Exemplary antisense sequences that target the SMN2 pre-mRNAand induce improved exon 7 inclusion are listed below as SEQ ID NOs:36-38. It is contemplated that selected modifications of these oligomersequences using the modified linkages and terminal groups describedherein would have improved properties compared to those known in theart. Furthermore, it is contemplated that any oligomer targeted tointron 7 of the SMN2 gene and incorporating the features of the presentinvention has the potential to induce exon 7 inclusion and provide atherapeutic benefit to SMA patients. Myotonic Dystrophy type 1 (DM1) andtype 2 (DM2) are dominantly inherited disorders caused by expression ofa toxic RNA leading to neuromuscular degeneration. DM1 and DM2 areassociated with long polyCUG and polyCCUG repeats in the 3′-UTR andintron 1 regions of the transcript dystrophia myotonica protein kinase(DMPK) and zinc finger protein 9 (ZNF9), respectively (see e.g.,WO2008/036406). While normal individuals have as many as 30 CTG repeats,DM1 patients carry a larger number of repeats ranging from 50 tothousands. The severity of the disease and the age of onset correlateswith the number of repeats. Patients with adult onsets show mildersymptoms and have less than 100 repeats, juvenile onset DM1 patientscarry as many as 500 repeats and congenital cases usually have around athousand CTG repeats. The expanded transcripts containing CUG repeatsform a secondary structure, accumulate in the nucleus in the form ofnuclear foci and sequester RNA-binding proteins (RNA-BP). Several RNA-BPhave been implicated in the disease, including muscleblind-like (MBNL)proteins and CUG-binding protein (CUGBP). MBNL proteins are homologousto Drosophila muscleblind (Mbl) proteins necessary for photoreceptor andmuscle differentiation. MBNL and CUGBP have been identified asantagonistic splicing regulators of transcripts affected in DM1 such ascardiac troponin T (cTNT), insulin receptor (IR) and muscle-specificchloride channel (ClC-1).

It is known in the art that antisense oligonucleotides targeted to theexpanded repeats of the DMPK gene can displace RNA-BP sequestration andreverse myotonia symptoms in an animal model of DM1 (WO2008/036406). Itis contemplated that oligomers incorporating features of the presentinvention would provide improved activity and therapeutic potential forDM1 and DM2 patients. Exemplary sequences targeted to the polyCUG andpolyCCUG repeats described above are listed below as SEQ ID NOs: 39-55and further described in U.S. application Ser. No. 13/101,942 which isincorporated herein in its entirety.

Additional embodiments of the present invention for treatingneuralmuscular disorders are anticipated and include oligomers designedto treat other DNA repeat instability genetic disorders. These diseasesinclude Huntington's disease, spino-cerebellar ataxia, X-linked spinaland bulbar muscular atrophy and spinocerebellar ataxia type 10 (SCA10)as described in WO2008/018795.

Experiments performed in support of the invention using the MDX mouse, amurine model for DMD, are described in Example 27.

TABLE 7 M23D sequences (SEQ ID NO: 15) that incorporate modifiedintersubunit linkages and/or 3′ and/or 5′ terminal groups PMO-X NGModification 5′ Sequence 3′ NG-10-0383 PMO EG3GGC CAA ACC TCG GCT TAC CTG triphenylacetyl AAA T NG-10-0325triphenylphos OH GGC CAA ACC FCG GCF TAC CFG triphenylphos AAA TNG-10-0272 PMO-farnesyl OH GGC CAA ACC TCG GCT TAC CTG farnesyl AAA TNG-10-0102 PMO OH GGC CAA ACC TCG GCT TAC CTG trityl AAA T NG-10-0330trimethoxybenzoyl EG3 GGC CAA ACC TCG GCT TAC CTG trimethoxybenzoylAAA T NG-10-0056 PMOplus 5′-pol EG3 GGC C⁺ A ⁺ A ⁺ ACC TCG GCT TAC HCTG AAA T NG-07-0064 PMO-3′-trityl H-Pip GGC CAA ACC TCG GCT TAC CTGtrityl AAA T NG-10-0382 PMO EG3 GGC CAA ACC TCG GCT TAC CTGtriphenylpropionyl AAA T NG-10-0278 PMOpyr EG3GGC CAA ACC pTCG GCpT pTAC H CpTG AAA pT NG-10-0210 PMOapn EG3 GGC C^(a)A ^(a) A ^(a) ACC TCG GCT TAC H CTG AAA T NG-10-0098 PMOpyr EG3GGC CAA ACC ^(p) TCG GC^(p) T TAC H C^(p) TG AAA T NG-10-0070 PMOapn EG3GGC CAA ACC ^(a) TCG GC^(a) T TAC H C^(a) TG AAA ^(a) T NG-10-0095PMOapn EG3 GGC CAA ACC ^(a) TCG GC^(a) T ^(a) TAC C^(a) T H G AAA ^(a) TNG-10-0317 PMO EG3 GGC CAA ACC TCG GCT TAC CTG farnesyl AAA T NG-10-0477PMO triMe Gly EG3 GGC CAA ACC FCG GCF TAC CFG trimethyl Glycine AAA FNG-10-0133 PMOapn OH GGC C^(a) AA ^(a) ACC ^(a) TCG GC^(a) T ^(a) TAC HC^(a) TG AAA ^(a) T NG-10-0387  PMO EG3 GGC CAA ACC TCG GCT TAC CTG2-OH, diphenylacet AAA T NG-10-0104  PMOguan EG3 GGC CAA ACC ^(g)TCG GC^(g) T TAC C^(g) T Δ^(g) G AAA T NG-10-0420  PMOplus methyl EG3GGC CAA ACC ^(m+) TCG GC^(m+) T TAC Trityl C^(m+) TG AAA ^(m+) TNG-10-0065  PMOtri EG3 GGC CAA ACC ^(t) TCG GC^(t) T TAC C^(t) T HG AAA T NG-10-0607  PMO-X EG3 GGC CAA ACC TCG GCT TAC CTG9-fluorene-carboxyl AAA T NG-10-0060  PMOcp EG3 GGC CAA ACC ^(cp)TCG GC^(cp) T TAC H C^(cp) T G AAA T NG-10-0162  PMO-COCH₂SH EG3GGC CAA ACC TCG GCT TAC CTG COCH₂SH AAA T NG-10-0328  diphenylacetyl EG3GGC CAA ACC TCG GCT TAC CTG diphenylacetyl AAA T NG-10-0134 PMOapnPMOtri OH GGC C^(a) AA ^(a) ACC ^(t) TCG GC^(t) T ^(t) TAC H C^(t)TG AAA ^(t) T NG-10-0386  PMO DPA GGC CAA ACC TCG GCT TAC CTG5′-diphenylac,3′- AAA T trity NG-07-0064  PMO-3′-trityl H-Pip GGC CAA ACC TCG GCT TAC CTG trityl AAA T NG-10-0059  PMOcp EG3GGC CAA ACC ^(cp) TCG GC^(cp) T ^(cp) TAC H C^(cp) T G AAA ^(cp) TNG-10-0135  PMOtri OH GGC CAA ACC ^(t) TCG GC^(t) T ^(t) TAC H C^(t)TG AAA ^(t) T NG-10-0168  PMOapn PMOcys OH GGC CAA ACC ^(a) TCG GC^(a)T ^(a) TAC H C^(a) TG AAA ^(SHc) T NG-10-0113  PMOapnPMOtri OHGGC CAA ACC ^(a) TCG GC^(t) T ^(t) TAC H C^(a) TG AAA ^(a) T NG-10-0385 PMO EG3 GGC CAA ACC TCG GCT TAC CTG diphenylphosphoryl AAA T NG-10-0279 PMO OH GGC CAA ACC TCG GCT TAC CTG geranyl AAA T NG-10-0055 PMOplus disp EG3 GGC C⁺ AA ⁺ ACC ⁺ TCG GC⁺ T TAC H C⁺ TG AAA TNG-10-0105  PMOsucc EG3 GGC CAA ACC ^(s) TCG GC^(s) T TAC C^(s) T Δ^(S)G AAA T NG-10-0805 PMO-X EG3 GGC CAA ACC ^(Etpip) TCG GC^(Etpip) T TAC HC^(Etpip) TG AAA ^(Etpip) T NG-10-0811 PMO-X EG3 GGC CAA ACC ^(pyrQMe)TCG GC^(pyrQMe) T H TAC C^(pyrQMe) TG AAA ^(pyrQMe) T NG-10-0057PMOplus 3′-pol EG3 GGC CAA ACC TCG GCT TAC C⁺ TG H ⁺ A ⁺ A ⁺ A TNG-10-0625 PMO-X EG3 GGC CAA ACC TCG GCT TAC CTG 5-carboxyfluoresceinAAA T NG-10-0804 dimer EG3 GGC CAA ACC TCG GCT TAC CTG dimerized AAA TNG-10-0066 PMOtri EG3 GGC CAA ACC ^(t) TCG GC^(t) T TAC C^(t) T HG AAA ^(t) T NG-10-0280 PMO disulfide EG3 GGC CAA ACC TCG GCT TAC CTGCOCH₂ CH₂SSPy AAA T NG-10-0212 PMOapn EG3 GGC CaAaA aACC aTCG GCaT HaTaAC CaTG aAaAaA aT NG-10-0156 3′-MeOtrityl EG3GGC CAA ACC TCG GCT TAC CTG MeO-Tr AAA T NG-10-0062 PMOhex EG3GGC CAA ACC ^(h) TCG GC^(h) T TAC C^(h) T H G AAA ^(h) T NG-11-0043PMO-X EG3 GGC CAA ACC TCG GCT TAC CTG guanidinyl AAA T NG-10-0206PMOplus EG3 GGC C+A+A +ACC +TCG GC+T H +T+AC C+TG +A+A+A +T NG-10-0383PMO EG3 GGC CAA ACC TCG GCT TAC CTG triphenylacetyl AAA T NG-10-0325triphenylphos OH GGC CAA ACC FCG GCF TAC CFG triphenylphos AAA TNG-10-0272 PMO-farnesyl OH GGC CAA ACC TCG GCT TAC CTG farnesyl AAA T*Dimerized indicates the oligomer is dimerized by a linkage linking the3′ ends of the two monomers. For example, the linkage may be-COCH₂CH₂-S-CH(CONH₂)CH₂-CO-NHCH₂CH₂CO- or any other suitable linkage.

11. Antibacterial Applications

The invention includes, in another embodiment, an antibacterialantisense oilgomer for use in treating a bacterial infection in amammalian host. In some embodiments, the oligomer comprises at least onelinkage of type (B) and/or at least one terminal modification (e.g., atleast one R²⁰) or combinations thereof, having between 10-20 bases and atargeting sequence of at least 10 contiguous bases complementary to atarget region of the infecting bacteria's mRNA for acyl carrier protein(acpP), gyrase A subunit (gyrA), ftsZ, ribosomal protein S10 (rpsJ),leuD, mgtC, pirG, pcaA, and cma1 genes, where the target region containsthe translational start codon of the bacterial mRNA, or a sequence thatis within 20 bases, in an upstream (i.e., 5′) or downstream (i.e., 3′)direction, of the translational start codon, and where the oligomerbinds to the mRNA to form a heteroduplex thereby to inhibit replicationof the bacteria.

Also included are conjugates of the oligomers where conjugated to theoligomers is an arginine-rich carrier protein coupled to theoligonucleotide at the peptide's carboxyl terminus, and preferablyrepresented by the peptide sequence (RXX)_(n)— or (RXR)_(b), where X isan uncharged amino acid selected from the group consisting of alanine,β-alanine, valine, leucine, isoleucine, serine, glycine threonine,phenyalanine, tryptophan, and 6-aminohexanoic acid, and n=2 to 4. Inexemplary embodiments, the carrier peptide has the sequence (RFF)_(n),(RFF)_(n)R, or (RXR)_(n) where n=2 to 4. The carrier peptide may belinked at its C-terminus to one end of the oligomer, e.g., the 3′ or5′-end, through a one- or two-amino acid linker, such as the linkerAhxβAla, where Ahx is 6-aminohexanoic acid and βAla is β-alanine. Thecarrier peptide has the ability, when conjugated to the 3′ or 5′-end ofthe oligonucleotide, to enhance the anti-bacterial activity of theoligonucleotide, as measured by inhibition in bacterial growth in vitroover an eight-hour period, by a factor of at least 10, and preferably10² or 10³. In a preferred embodiment, the carrier peptide has thesequence (RAhxR)_(n)—, where n=4.

12. Modulating Nuclear Hormone Receptors

In another embodiment the present invention relates to compositions andmethods for modulating expression of nuclear hormone receptors (NHR)from the nuclear hormone receptor superfamily (NHRSF), mainly bycontrolling or altering the splicing of pre-mRNA that codes for thereceptors. Examples of particular NHRs include glucocorticoid receptor(GR), progesterone receptor (PR) and androgen receptor (AR). In certainembodiments, the antisense oligonucleotides and agents described hereinlead to increased expression of ligand-independent or other selectedforms of the receptors, and decreased expression of their inactiveforms.

Embodiments of the present invention include oligomers andoligonucleotide analogs, for example oligomers comprising at least onelinkage of type (B) and/or at least one terminal modification (e.g., atleast one R²⁰) or combinations thereof, that are complementary toselected exonic or intronic sequences of an NHR, including the“ligand-binding exons” and/or adjacent introns of a NHRSF pre-mRNA,among other NHR-domains described herein. The term “ligand-bindingexons” refers to exon(s) that are present in the wild-type mRNA but areremoved from the primary transcript (the “pre-mRNA”) to make aligand-independent form of the mRNA. In certain embodiments,complementarity can be based on sequences in the sequence of pre-mRNAthat spans a splice site, which includes, but is not limited to,complementarity based on sequences that span an exon-intron junction. Inother embodiments, complementarity can be based solely on the sequenceof the intron. In other embodiments, complementarity can be based solelyon the sequence of the exon. (See, e.g., U.S. application Ser. No.13/046,356, which is incorporated herein by reference.)

NHR modulators may be useful in treating NHR-associated diseases,including diseases associated with the expression products of geneswhose transcription is stimulated or repressed by NHRs. For instance,modulators of NHRs that inhibit AP-1 and/or NF-κB can be useful in thetreatment of inflammatory and immune diseases and disorders such asosteoarthritis, rheumatoid arthritis, multiple sclerosis, asthma,inflammatory bowel disease, transplant rejection, and graft vs. hostdisease, among others described herein and known in the art. Compoundsthat antagonize transactivation can be useful in treating metabolicdiseases associated with increased levels of glucocorticoid, such asdiabetes, osteoporosis and glaucoma, among others. Also, compounds thatagonize transactivation can be useful in treating metabolic diseasesassociated with a deficiency in glucocorticoid, such as Addison'sdisease and others.

Embodiments of the present invention include methods of modulatingnuclear NHR activity or expression in a cell, comprising contacting thecell with an antisense oligomer composed of morpholino subunits linkedby phosphorus-containing intersubunit linkages joining a morpholinonitrogen of one subunit to a 5′ exocyclic carbon of an adjacent subunit,wherein the oligonucleotide contains between 10-40 bases and a targetingsequence of at least 10 contiguous bases complementary to a targetsequence, wherein the target sequence is a pre-mRNA transcript of theNHR, thereby modulating activity or expression of the NHR. In certainembodiments, the oligomer alters splicing of the pre-mRNA transcript andincreases expression of a variant of the NHR. In some embodiments, theoligomer induces full or partial exon-skipping of one or more exons ofthe pre-mRNA transcript. In certain embodiments, the one or more exonsencode at least a portion of a ligand-binding domain of the NHR, and thevariant is a ligand independent form of the NHR. In certain embodiments,the one or more exons encode at least a portion of a transactivationdomain of the NHR, and the variant has reduced transcriptionalactivation activity. In certain embodiments, the one or more exonsencode at least a portion of a DNA-binding domain of the NHR. In certainembodiments, the one or more exons encode at least a portion of anN-terminal activation domain of the NHR. In certain embodiments, the oneor more exons encode at least a portion of a carboxy-terminal domain ofthe NHR. In specific embodiments, the variant binds to NF-KB, AP-1, orboth, and reduces transcription of one or more of their pro-inflammatorytarget genes.

In certain embodiments, the oligomer agonizes a transactivationaltranscriptional activity of the NHR. In other embodiments, the oligomerantagonizes a transactivational transcriptional activity of the NHR. Incertain embodiments, the oligomer agonizes a transrepression activity ofthe NHR. In other embodiments, the oligomer antagonizes atransrepression activity of the NHR. In specific embodiments, theoligomer antagonizes a transactivational transcriptional activity of theNHR and agonizes a transrepression activity of the NHR. (See, e.g., U.S.Appn. No. 61/313,652, which is incorporated herein by reference.)

EXAMPLES

Unless otherwise noted, all chemicals were obtained fromSigma-Aldrich-Fluka. Benzoyl adenosine, benzoyl cytidine, andphenylacetyl guanosine were obtained from Carbosynth Limited, UK.

Synthesis of PMO, PMO+, PPMO and PMO containing further linkagemodifications as described herein was done using methods known in theart and described in pending U.S. application Ser. Nos. 12/271,036 and12/271,040 and PCT publication number WO/2009/064471, which are herebyincorporated by reference in their entirety.

PMO with a 3′ trityl modification are synthesized essentially asdescribed in PCT publication number WO/2009/064471 with the exceptionthat the detritylation step is omitted.

Example 1 tert-butyl4-(2,2,2-trifluoroacetamido)piperidine-1-carboxylate

To a suspension of tert-butyl 4-aminopiperidine-1-carboxylate (48.7 g,0.243 mol) and DIPEA (130 mL, 0.749 mol) in DCM (250 mL) was added ethyltrifluoroacetate (35.6 mL, 0.300 mol) dropwise while stirring. After 20hours, the solution was washed with citric acid solution (200 mL×3, 10%w/v aq) and sodium bicarbonate solution (200 mL×3, conc aq), dried(MgSO₄), and filtered through silica (24 g). The silica was washed withDCM and the combined eluant was partially concentrated (100 mL), andused directly in the next step. APCI/MS calcd. for C₁₂H₁₉F₃N₂O₃ 296.1,found m/z=294.9 (M−1).

Example 2 2,2,2-trifluoro-N-(piperidin-4-yl)acetamide hydrochloride

To a stirred DCM solution of the title compound of Example 1 (100 mL)was added dropwise a solution of hydrogen chloride (250 mL, 1.0 mol) in1,4-dioxane (4 M). Stirring was continued for 6 hours, then thesuspension was filtered, and the solid washed with diethyl ether (500mL) to afford the title compound (54.2 g, 96% yield) as a white solid.APCI/MS calcd. for C₇H₁₁F₃N₂O 196.1, found m/z=196.9 (M+1).

Example 3 (4-(2,2,2-trifluoroacetamido)piperidin-1-yl)phosphonicdichloride

To a cooled (ice/water bath) suspension of the title compound of Example2 (54.2 g, 0.233 mol) in DCM (250 mL) was added dropwise phosphorusoxychloride (23.9 mL, 0.256 mol) and DIPEA (121.7 mL, 0.699 mol) andstirred. After 15 minutes, the bath was removed and with continuedstirring the mixture allowed to warm to ambient temperature. After 1hour, the mixture was partially concentrated (100 mL), the suspensionfiltered, and the solid washed with diethyl ether to afford the titlecompound (43.8 g, 60% yield) as a white solid. The elutant was partiallyconcentrated (100 mL), the resulting suspension filtered, and the solidwashed with diethyl ether to afford additional title compound (6.5 g, 9%yield). ESI/MS calcd. for 1-(4-nitrophenyl)piperazine derivativeC₁₇H₂₂ClF₃N₅O₄P 483.1, found m/z=482.1 (M−1).

Example 4((2S,6S)-6-((r)-5-methyl-2,6-dioxo-1,2,3,6-tetrahydropyridin-3-yl-4-tritylmorpholin-2-yl)methyl(4-(2,2,2-trifluoroacetamido)piperidin-1-yl)phosphonochloridate

To a stirred, cooled (ice/water bath) solution of the title compound ofExample 3 (29.2 g, 93.3 mmol) in DCM (100 mL) was added dropwise over 10minutes a DCM solution (100 mL) of Mo(Tr)T # (22.6 g, 46.7 mmol),2,6-Lutidine (21.7 mL, 187 mmol), and 4-(dimethylamino)pyridine (1.14 g,9.33 mmol). The bath was allowed to warm to ambient temperature. After15 hours, the solution was washed with a citric acid solution (200 mL×3,10% w/v aq), dried (MgSO₄), concentrated, and the crude oil was loadeddirectly onto column. Chromatography [SiO₂ column (120 g), hexanes/EtOAceluant (gradient 1:1 to 0:1), repeated×3] fractions were concentrated toprovide the title compound (27.2 g, 77% yield) as a white solid. ESI/MScalcd. for the 1-(4-nitrophenyl)piperazine derivative C₄₆H₅₀F₃N₈O₈P930.3, found m/z=929.5 (M−1).

Example 5((2S,6R)-6-(6-benzamido-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl(4-(2,2,2-trifluoroacetamido)piperidin-1-yl)phosphonochloridate

The title compound was synthesized in a manner analogous to thatdescribed in Example 4 to afford the title compound (15.4 g, 66% yield)as a white solid. ESI/MS calcd. for 1-(4-nitrophenyl)piperazinederivative C₅₃H₅₃F₃N₁₁O₇P 1043.4, found m/z=1042.5 (M−1).

Example 6 (R)-methyl(1-phenylethyl)phosphoramidic dichloride

To a cooled (ice/water bath) solution of phosphorus oxychloride (2.83mL, 30.3 mmol) in DCM (30 mL) was added sequentially, dropwise, and withstirring 2,6-lutidine (7.06 mL, 60.6 mmol) and a DCM solution of(R)-(+)-N,a-dimethylbenzylamine (3.73 g, 27.6 mmol). After 5 minutes,the bath was removed and reaction mixture allowed to warm to ambienttemperature. After 1 hour, the reaction solution was washed with acitric acid solution (50 mL×3, 10% w/v aq), dried (MgSO₄), filteredthrough SiO₂ and concentrated to provide the title compound (3.80 g) asa white foam. ESI/MS calcd. for 1-(4-nitrophenyl)piperazine derivativeC₁₉H₂₅N₄O₄P 404.2, found m/z=403.1 (M−1).

Example 7 (S)-methyl(1-phenylethyl)phosphoramidic dichloride

The title compound was synthesized in a manner analogous to thatdescribed in Example 6 to afford the title compound (3.95 g) as a whitefoam. ESI/MS calcd. for 1-(4-nitrophenyl)piperazine derivativeC₁₉H₂₅N₄O₄P 404.2, found m/z=403.1 (M−1).

Example 8((2S,6R)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methylmethyl((R)-1-phenylethyl)phosphoramidochloridate

The title compound was synthesized in a manner analogous to thatdescribed in Example 4 to afford the title chlorophosphoroamidate (4.46g, 28% yield) as a white solid. ESI/MS calcd. for C₃₈H₄0ClN₄O₅P 698.2,found m/z=697.3 (M−1).

Example 9((2S,6R)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methylmethyl((S)-1-phenylethyl)phosphoramidochloridate

The title compound was synthesized in a manner analogous to thatdescribed in Example 4 to afford the title chlorophosphoroamidate (4.65g, 23% yield) as a white solid. ESI/MS calcd. for C₃₈H₄0ClN₄O₅P 698.2,found m/z=697.3 (M−1).

Example 10 (4-(pyrrolidin-1-yl)piperidin-1-yl)phosphonic dichloridehydrochloride

To a cooled (ice/water bath) solution of phosphorus oxychloride (5.70mL, 55.6 mmol) in DCM (30 mL) was added 2,6-lutidine (19.4 mL, 167 mmol)and a DCM solution (30 mL) of 4-(1-pyrrolidinyl)-piperidine (8.58 g,55.6 mmol) and stirred for 1 hour. The suspension was filtered and solidwashed with excess diethyl ether to afford the title pyrrolidine (17.7g, 91% yield) as a white solid. ESI/MS calcd. for1-(4-nitrophenyl)piperazine derivative C₁₉H₃₀N₅O₄P 423.2, foundm/z=422.2 (M−1).

Example 11((2S,6R)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methyl(4-(pyrrolidin-1-yl)piperidin-1-yl)phosphonochloridatehydrochloride

To a stirred, cooled (ice/water bath) solution of thedichlorophosphoramidate 8 (17.7 g, 50.6 mmol) in DCM (100 mL) was addeda DCM solution (100 mL) of Mo(Tr)T # (24.5 g, 50.6 mmol), 2,6-Lutidine(17.7 mL, 152 mmol), and 1-methylimidazole (0.401 mL, 5.06 mmol)dropwise over 10 minutes. The bath was allowed to warm to ambienttemperature as suspension was stirred. After 6 hours, the suspension waspoured onto diethyl ether (1 L), stirred 15 minutes, filtered and solidwashed with additional ether to afford a white solid (45.4 g). The crudeproduct was purified by chromatography [SiO₂ column (120 gram), DCM/MeOHeluant (gradient 1:0 to 6:4)], and the combined fractions were pouredonto diethyl ether (2.5 L), stirred 15 min, filtered, and the resultingsolid washed with additional ether to afford the title compound (23.1 g,60% yield) as a white solid. ESI/MS calcd. for1-(4-nitrophenyl)piperazine derivative C₄₈H₅₇N₈O₇P 888.4, foundm/z=887.6 (M−1).

Example 12 3-(tert-butyldisulfanyl)-2-(isobutoxycarbonylamino)propanoicacid

To S-tert-butylmercapto-L-cysteine (10 g, 47.8 mmol) in CH₃CN (40 mL)was added K₂CO₃ (16.5 g, 119.5 mmol) in H₂O (20 mL). After stirring for15 minutes, iso-butyl chloroformate (9.4 mL, 72 mmol) was injectedslowly. The reaction was allowed to run for 3 hours. The white solid wasfiltered through Celite; the filtrate was concentrated to remove CH₃CN.The residue was dissolved in ethyl acetate (200 mL), washed with 1N HCl(40 ml×3), brine (40×1), dried over Na₂SO₄. Desired product (2) wasobtained after chromatography (5% MeOH/DCM).

Example 13 tert-butyl4-(3-(tert-butyldisulfanyl)-2-(isobutoxycarbonylamino)propanamido)piperidine-1-carboxylate

To the acid (compound 2 from Example 12, 6.98 g, 22.6 mmol) in DMF (50ml was added HATU (8.58 g, 22.6 mmol). After 30 min, Hunig base (4.71ml, 27.1 mmol) and 1-Boc-4-amino piperidine (5.43 g, 27.1 mmol) wereadded to the mixture. The reaction was continued stirring at RT foranother 3 h. DMF was removed at high vacuum, the crude residue wasdissolved in EtAc (300 ml), washed with H₂O (50 ml×3). The final product(3) was obtained after ISCO purification (5% MeOH/DCM).

Example 14 isobutyl3-(tert-butyldisulfanyl)-1-oxo-1-(piperidin-4-ylamino)propan-2-ylcarbamate

To compound 3 prepared in Example 13 (7.085 g, 18.12 mmol) was added 30ml of 4M HCl/Dioxane. The reaction was completed after 2 h at RT. TheHCl salt (4) was used for the next step without further purification.

Example 15 isobutyl3-(tert-butyldisulfanyl)-1-(1-(dichlorophosphoryl)piperidin-4-ylamino)-1-oxopropan-2-ylcarbamate

To compound 4 prepared in Example 15 (7.746 g, 18.12 mmol) in DCM (200ml) at −78° C. was slowly injected POCl₃ (1.69 ml, 18.12 mmol) under Ar,followed by the addition of Et₃N (7.58 ml, 54.36 mmol). The reaction wasstirred at RT for 5 h, concentrated to remove excess base and solvent.The product (5) was given as white solid after ISCO purification (50%EtAc/Hexane).

Example 16 isobutyl3-(tert-butyldisulfanyl)-1-(1-(chloro(((2S,6R)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methoxy)phosphoryl)piperidin-4-ylamino)-1-oxopropan-2-ylcarbamate

To1-((2R,6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione(moT(Tr)) (5.576 g, 10.98 mmol) in DCM (100 ml) at 0° C., was addedlutidine (1.92 ml, 16.47 mmol) and DMAP (669 mg, 5.5 mmol), followed bythe addition of 4 (6.13 g, 12.08 mmol). The reaction was left stirringat RT for 18 h. The desired product (6) was obtained after ISCOpurification (50% EtAc/Hexane).

Example 17((2S,6R)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methylhexyl(methyl)phosphoramidochloridate

A DCM (80ml) solution of N-hydroxylmethylamine (4.85 ml, 32 mmol) wascooled down to −78° C. under N2. A solution of phosphoryl chloride (2.98ml, 32 mmol) in DCM (10 ml), followed by a solution of Et₃N (4.46 ml, 32mmol) in DCM (10 ml), was added slowly. The stirring was continued whilethe reaction was allowed to warm to RT overnight. The desired product(1) was given as clear oil after ISCO purification (20% EtAc/Hexane).

To moT(Tr) (5.10 g, 10.54 mmol) in DCM (100 ml) at 0° C., was addedlutidine (3.68 ml, 31.6 mmol) and DMAP (642 mg, 5.27 mmol), followed bythe addition of 1 (4.89 g, 21.08 mmol). The reaction was left stirringat RT for 18 h. The desired product (2) was obtained after ISCOpurification (50% EtOAc/Hexane).

Example 18((2S,6R)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methyldodecyl(methyl)phosphoramidochloridate

The title compound was prepared according to the general proceduresdescribed in Examples 6 and 8.

Example 19((2S,6R)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methylmorpholinophosphonochloridate

The title compound was prepared according to the general proceduresdescribed in Examples 6 and 8.

Example 20((2S,6R)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methyl(S)-2-(methoxymethyl)pyrrolidin-1-ylphosphonochloridate

The title compound was prepared according to the general proceduresdescribed in Examples 6 and 8.

Example 21((2S,6R)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methyl4-(3,4,5-trimethoxybenzamido)piperidin-1-ylphosphonochloridate

To 1-Boc-4-piperidine (1 g, 5 mmol) in DCM (20 ml) was added Hunig base(1.74 ml, 10 mmol), followed by the addition of 3,4,5-trimethoxybenzoylchloride (1.38 g, 6 mmol). The reaction was run at RT for 3 h,concentrated to remove solvent and excess base. The residue wasdissolved in EtAc (100 ml), washed with 0.05N HCl (3×15 ml), sat. NaHCO₃(2×15 ml), dried over Na₂SO₄. Product (1) was obtained after ISCOpurification (5% MeOH/DCM).

To 7 was added 15 ml of 4N HCl/Dioxane, reaction was terminated after 4h. 8 was obtained as white solid.

A DCM (20 ml) solution of 8 (1.23 g, 4.18 mmol) was cooled down to −78°C. under N₂. A solution of phosphoryl chloride (0.39 ml, 4.18 mmol) inDCM (2 ml), followed by a solution of Et₃N (0.583 ml, 4.18 mmol) in DCM(2 ml), was added slowly. The stirring was continued while the reactionwas allowed to warm to RT overnight. The desired product (9) wasobtained after ISCO purification (50% EtAc/Hexane).

To moT(Tr) (1.933 g, 4.0 mmol) in DCM (20 ml) at 0° C., was addedlutidine (0.93 ml, 8 mmol) and DMAP (49 mg, 0.4 mmol), followed by theaddition of 9 (1.647 g, 4 mmol). The reaction was left stirring at RTfor 18 h. The desired product (10) was obtained after ISCO purification(50% EtAc/Hexane).

Example 22 Synthesis of Cyclophosphoramide Containing Subunit (^(CP)T)

The moT subunit (25 g) was suspended in DCM (175 ml) and NMI(N-methylimidazole, 5.94 g, 1.4 eq.) was added to obtain a clearsolution. Tosyl chloride was added to the reaction mixture, and thereaction progress was monitored by TLC until done (about 2 hours). Anaqueous workup was performed by washing with 0.5 M citric acid buffer(pH=5), followed by brine. The organic layer was separated and driedover Na2SO4. Solvent was removed with a rotavaporator to obtain thecrude product which was used in the next step without furtherpurification.

The moT Tosylate prepared above was mixed with propanolamine (1 g/10ml). The reaction mixture was then placed in an oven at 45° C. overnightfollowed by dilution with DCM (10 ml). An aqueous workup was performedby washing with 0.5 M citric acid buffer (pH=5), followed by brine. Theorganic layer was separated and dried over Na₂SO₄. Solvent was removedwith a rotavaporator to obtain the crude product. The curde product wasanalyzed by NMR and HPLC and determined to be ready for the next stepwithout further purification.

The crude product was dissolved in DCM (2.5 ml DCM/g, 1 eq.) and mixedwith DIEA (3 eq.). This solution was cooled with dry ice-acetone andPOCl₃ was added dropwise (1.5 eq.). The resultant mixture was stirred atroom temperature overnight. An aqueous workup was performed by washingwith 0.5 M citric acid buffer (pH=5), followed by brine. The organiclayer was separated and dried over Na₂SO₄. Solvent was removed with arotavaporator to obtain the crude product as a yellowish solid. Thecrude product was purified by silica gel chromatography (crudeproduct/silica=1 to 5 ratio, gradient DCM to 50% EA/DCM), and fractionswere pooled according to TLC analysis. Solvent was removed to obtain thedesired product as a mixture of diastereomers. The purified product wasanalyzed by HPLC (NPP quench) and NMR (H-1 and P-31).

The diastereomeric mixture was separated according to the followingprocedure. The mixture (2.6 g) was dissolved in DCM. This sample wasloaded on a RediSepRf column (80 g normal phase made by Teledyne Isco)and eluted with 10% EA/DCM to 50% EA/DCM over 20 minutes. Fractions werecollected and analyzed by TLC. Fractions were pooled according to TLCanalysis, and solvent was removed with a rotavaporator at roomtemperature. The diastereomeric ratio of ther pooled fractions wasdetermined by P-31 NMR and NPP-TFA analysis. If needed, the aboveprocedure was repeated until the diastereomeric ratio reached 97%.

Example 23 Global Cholic Acid Modification of PMOplus

The succinimide activated cholic acid derivative was prepared accordingto the following procedure. Cholic acid (12 g, 29.4 mmol),N-hydroxysuccinimide (4.0 g, 34.8 mmol), EDCI (5.6 g, 29.3 mmol), andDMAP (1 g, 8.2 mmol) were charged to a round bottom flask. DCM (400 ml)and THF (40 ml) were added to dissolve. The reaction mixture was stirredat room temperature overnight. Water (400 ml) was then added to thereaction mixture, the organic layer separated and washed with water(2×400 ml), followed by sat. NaHCO₃ (300 ml) and brine (300 ml). Theorganic layer was then dried over Na₂SO₄. Solvent was removed withrotavaporator to obtain a white solid. The curde product was dissolvedin chloroform (100 ml) and precipitated into heptane (1000 ml). Thesolid was collected by filtration, analyzed by HPLC and NMR and usedwithout further purification.

An appropriate amount of PMOplus (20 mg, 2.8 μmol) was weighed into avial (4 ml) and dissolved in DMSO (500 ul). The activated cholate ester(13 mg, 25 μmol) was added to the reaction mixture according to theratio of two equivalent of active ester per modification site followedby stirring at room temperature overnight. Reaction progress wasdetermined by MALDI and HPLC (C-18 or SAX).

After the reaction was complete (as determined by disappearance ofstarting PMOplus), 1 ml of concentrated ammonia was added to thereaction mixture once the reaction is complete. The reaction vial wasthen placed in an oven (45° C.) overnight (18 hours) followed by coolingto room temperature and dilution with 1% ammonia in water (10 ml). Thissample was loaded on to an SPE column (2 cm), and the vial rinsed with1% ammonia solution (2×2 ml). The SPE column was washed with 1% ammoniain water (3×6 ml), and the product eluted with 45% acetonitrile in 1%ammonia in water (6 ml). Fractions containing oligomer were identifiedby UV optical density measurement. Product was isolated bylyophilization. Purity and identity were determined by MALDI and HPLC(C-18 and/or SAX).

This same procedure is applicable to deoxycholic acid activation andconjugation to a PMO⁺.

Example 24 Global Guanidinylation of PMOplus

An appropriate amount of PMOplus (25 mg, 2.8 μmol) was weighed into avial (6 ml). 1H-Pyrozole-1-carboxamidine chloride (15 mg, 102 μmol) andpotassium carbonate (20 mg, 0.15 mmol) were added to the vial. Water wasadded (500 ul), and the reaction mixture was stirred at room temperatureovernight (about 18 hours). Reaction completion was determined by MALDI.

Once complete, the reaction was diluted with 1% ammonia in water (10 ml)and loaded on to an SPE column (2 cm). The vial was rinsed with 1%ammonia solution (2×2 ml), and the SPE column was washed with 1% ammoniain water (3×6 ml). Product was eluted with 45% acetonitrile in 1%ammonia in water (6 ml). Fractions containing oligomer were identifiedby UV optical density measurement. Product was isolated bylyophilization. Purity and identity were determined by MALDI and HPLC(C-18 and/or SAX).

Example 25 Global Thioacetyl Modification of PMOplus (M23D)

An appropriate amount of PMOplus (20 mg, 2.3 μmol) was weighed in to avial (4 ml) and dissolved in DMSO (500 ul).N-succinimidyl-S-acetylthioacetate (SATA) (7 mg, 28 μmol) was added tothe reaction mixture, and it was allowed to stir at room temperatureovernight. Reaction progress was monitored by MALDI and HPLC.

Once complete, 1% ammonia in water was added to the reaction mixture,and it was stirred at room temperature for 2 hours. This solution wasloaded on to an SPE column (2 cm), The vial was rinsed with 1% ammoniasolution (2×2 ml), and the SPE column was washed with 1% ammonia inwater (3×6 ml). Product was eluted with 45% acetonitrile in 1% ammoniain water (6 ml). Fractions containing oligomer were identified by UVoptical density measurement. Product was isolated by lyophilization.Purity and identity were determined by MALDI and HPLC (C-18 and/or SAX).

Example 26 Global Succinic Acid Modification of PMOplus

An appropriate amount of PMOplus (32 mg, 3.7 μmol) was weighed in to avial (4 ml) and dissolved in DMSO (500 ul). N-ethyl morpholino (12 mg,100 μmol) and succinic anhydride (10 mg, 100 μmol) were added to thereaction mixture, and it was allowed to stir at room temperatureovernight. Reaction progress was monitored by MALDI and HPLC.

Once complete, 1% ammonia in water was added to the reaction mixture,and it was stirred at room temperature for 2 hours. This solution wasloaded on to an SPE column (2 cm), The vial was rinsed with 1% ammoniasolution (2×2 ml), and the SPE column was washed with 1% ammonia inwater (3×6 ml). Product was eluted with 45% acetonitrile in 1% ammoniain water (6 ml). Fractions containing oligomer were identified by UVoptical density measurement. Product was isolated by lyophilization.Purity and identity were determined by MALDI and HPLC (C-18 and/or SAX).

The above procedure is applicable to glutartic acid (glutaric anhydride)and tetramethyleneglutaric acid (tetramethyleneglutaric anhydride)modification of PMOplus as well.

Example 27 Treatment of Mdx Mice with Exemplary PMO Oligomers of theInvention

The MDX mouse is an accepted and well-characterized animal model forDuchene muscular dystrophy (DMD) containing a mutation in exon 23 of thedystrophin gene. The M23D antisense sequence (SEQ ID NO:15) is known toinduce exon 23 skipping and restoration of functional dystrophinexpression. MDX mice were dosed once (50 mg/kg) by tail vein injectionwith either M23D PMO+ oligomers (NG-09-0711, NG-10-0055, NG-10-0056) ortwo PMO compounds containing either the 4-aminopiperidinyl linkage(NG-10-0070 containing the PMO^(apn) linkage described above and shownin FIG. 2) and the 4-succinamidopiperazinyl linkage (NG-10-0105containing the PMO^(suc) linkage described above and shown in FIG. 2). Apeptide-conjugated PMO (PPMO) was used as a positive control in theexperiment (AVI-5225; SEQ ID NO: 16). All tested oligomer has the sameantisense sequence, but varied by type of linkage or a peptide (in thecase of AVI-5225, see Table 8)). One week post-injection, the MDX micewere sacrificed and RNA was extracted from various muscle tissues.End-point PCR was used to determine the relative abundance of dystrophinmRNA containing exon 23 and mRNA lacking exon 23 due toantisense-induced exon skipping. Percent exon 23 skipping is a measureof antisense activity in vivo. FIG. 5 shows the results from thequadriceps one week post-treatment. NG-10-0070 containing four cationic4-aminopiperidinyl linkages shows a two-fold increase in activitycompared to any of the PMO+ compounds (NG-10-0055, -0056 and -0057). TheNG-10-0105 compound containing four anionic 4-succinamidopiperazinyllinkages was equally active compared to the PMO+ oligomers. As expectedthe AVI-5225 PPMO (peptide conjugated) compound was most effective dueto the cell penetrating delivery peptide. The vehicle and WT C57(wild-type mice) treatments were negative controls and did not expressexon 23 skipped dystrophin mRNA.

TABLE 8 Sequences of Example 27 SEQ ID Name Sequence (5′ to 3′) NO M23DGGCCAAACCTCGGCTTACCTGAAAT 15 NG-09-0711 GGC CAA ACC +TCG GC+T TAC C+TGN/A AAA +T NG-10-0055 GGC C⁺AA ⁺ACC ⁺TCG GC⁺T TAC C⁺TG N/A AAA TNG-10-0056 GGC C⁺A⁺A ⁺ACC TCG GCT TAC CTG N/A AAA T NG-10-0057GGC CAA ACC TCG GCT TAC C⁺TG N/A ⁺A⁺A⁺A T NG-10-0070GGC CAA ACC ^(apn)TCG GC^(apn)T TAC C^(apn)TG N/A AAA ^(apn)T NG-10-0105GGC CAA ACC ^(suc)TCG GC^(suc)T TAC C^(suc)TG N/A AAA T AVI-5225GGCCAAACCTCGGCTTACCTGAAAT- 16 RAhxRRBRRAhxRRBRAhxB

Additional experiments in support of the invention were performed usinga wider range of modified intersubunit linkages within the M23D PMO andused in the MDX mouse model as described above. A subset of theoligomers with the linkages are listed as above in Table 7. FIG. 6 showsthe results from this expanded screen and shows the M23D oligomers withthe highest activity are NG-10-0070, NG-10-0104, NG-10-0095 andNG-10-0133 comprising linkages b10, b54, b10 and b10, respectively (inFIG. 6, the labels on the x axis correspond to the last 3 digits of thecompound ID#). The MDX mice received a single injection intravenously ata 50 mg/kg dose. Other active compounds shown in FIG. 6 are M23D PMOcomprising terminal modifications and are described above in Table 6.All the compounds were compared to a PMO without any intersubunit orterminal modifications (SEQ ID NO:15).

Additional experiments in support of the invention used an even greaterexpansion of compounds with intersubunit and terminal linkages.Intersubunit linkage modifications are shown above in Table 9. Resultsusing those compounds are shown below in Table 9. The results areordered with the most active compounds at the top of the table.

TABLE 9 Exon 23 skipping in quadricep and diaphram tissue from MDX micetreated with PMO-X compounds of the invention Dose Exon skip % NG #PMO-X modification mg/kg Quads Diaph NG-10-0383 PMO 30 61 20 NG-10-0325triphenylphos 30 54 46 NG-10-0272 PMO-farnesyl 30 48 14 NG-10-0102 PMO30 44 23 NG-10-0330 trimethoxybenzoyl 30 40 7 NG-10-0056 PMOplus 5′-pol23 40 13 NG-07-0064 PMO-3′-trityl 30 37 24 NG-10-0382 PMO 30 36 18NG-10-0278 PMOpyr 26 35 29 NG-10-0210 PMOapn 31 34 19 NG-10-0098 PMOpyr30 31 19 NG-10-0070 PMOapn 30 30 10 NG-10-0095 PMOapn 30 30 11NG-10-0317 PMO 30 30 17 NG-10-0477 PMO triMe Gly 30 28 32 NG-10-0133PMOapn 30 28 17 NG-10-0387 PMO 30 28 25 NG-10-0104 PMOguan 30 27 14NG-10-0420 PMOplus methyl 29 27 25 NG-10-0065 PMOtri 30 26 2 NG-10-0607PMO-X 30 25 19 NG-10-0060 PMOcp 30 25 6 NG-10-0162 PMO-COCH₂SH 30 25 8NG-10-0328 diphenylacetyl 30 25 20 NG-10-0134 PMOapnPMOtri 30 23 2NG-10-0386 PMO 30 22 11 NG-07-0064 PMO-3′-trityl 30 22 23 NG-10-0059PMOcp 30 22 9 NG-10-0135 PMOtri 30 21 19 NG-10-0168 PMOapn PMOcys 30 216 NG-10-0113 PMOapnPMOtri 30 20 20 NG-10-0385 PMO 30 20 32 NG-10-0279PMO 30 19 22 NG-10-0055 PMOplus disp 30 17 11 NG-10-0105 PMOsucc 30 16 4NG-10-0805 PMO-X 30 16 21 NG-10-0811 PMO-X 32 16 6 NG-10-0057 PMOplus3′-pol 30 15 16 NG-10-0625 PMO-X 28 15 11 NG-10-0804 Dimer 35 15 11NG-10-0066 PMOtri 30 12 1 NG-10-0280 PMO disulfide 30 12 14 NG-10-0212PMOapn 20 11 15 NG-10-0156 3′-MeOtrityl 30 10 22 NG-10-0062 PMOhex 30 910 NG-11-0043 PMO-X 30 9 16 NG-10-0206 PMOplus 31 8 10

Example 28 Treatment of Transgenic eGFP Mice with Exemplary PMOOligomers of the Invention

Experiments in support of the invention used an eGFP-based assay for invivo antisense activity and was used to evaluate oligomers comprisingthe modified intersubunit linkages of the invention. The transgenic eGFPmouse model in which the eGFP-654 transgene, is expressed uniformlythroughout the body has been described (Sazani, Gemignani et al. 2002).This model uses a splicing assay for activity in which the modifiedoligomers of the present invention block aberrant splicing and restorecorrect splicing of the modified enhanced green fluorescent protein(eGFP) pre-mRNA. In this approach, antisense activity of each oligomeris directly proportional to up-regulation of the eGFP reporter. As aresult, the functional effects of the same oligomer can be monitored inalmost every tissue. This is in contrast to oligomers targeted to geneswhose expression is restricted to or is phenotypically relevant in onlycertain tissues. In the eGFP-654 mice, the pre-mRNA was readilydetectable in all tissues although smaller amounts were found in thebone marrow, skin and brain. The level of translated eGFP isproportional to the potency of the antisense oligomers and theirconcentration at the site of action. RT-PCR of total RNA isolated fromvarious tissues showed expression of eGFP-654 transcript in all tissuessurveyed.

Tissues from eGFP-654 mice (n=6) treated with compound ranging from 5 to150 mg/kg were collected 8 days post-dosing and frozen at −80° C.Tissues were thawed immediately prior to imaging on a GE Typhoon Trio,misted with PBS, and arrayed directly on the glass platen of thescanner. 50 micron scans to collect eGFP fluorescence were performedusing the 488 nm excitation laser and 520 nm BP 40 emission filter withthe focal plane at the platen surface. Tissue scans were analyzed usingImageQuant to determine average fluorescence across each tissue. Tissuefluorescence from 3-5 mice treated with vehicle only were averaged toyield an intrinsic background fluorescence measurement for each tissuetype. Fold-fluorescence values of the corresponding tissues fromcompound-treated mice were calculated as the fraction of the vehicletissue fluorescence. FIGS. 7B-C show the tissue specific activity in theeGFP-654 mouse model of two PMO containing exemplary intersubunitlinkages of the invention, NG-10-0110 and NG-10-0323-, containinglinkages b54 and b11, respectively. All of the oligomers tested arederived from the eGFP654 sequence (SEQ ID NO: 17). For comparison,results using a PMO having the same sequence, but lacking anyintersubunit modifications is shown in FIG. 7A. NG-10-0110 (SEQ IDNO:17) had high activity in quadriceps and poor activity in liver (FIG.7B) whereas NG-10-0323 had improved liver activity and muscle delivery(FIG. 7C).

Additional examples in support of the invention included experimentsusing eGFP (SEQ ID NO:17) oligomers modified using the linkages andterminal groups of the invention. As shown in FIGS. 11 and 12, comparedto PMO and PMOplus oligomers, several modified oligomers showed improvedeGFP splice-correction activity in various tissues from mice treated asdescribed above.

The specific PMO-X modifications of the compounds described in thisexample are shown below in Table 10.

TABLE 10 Sequences Used in Example 28 Showing Linkage Type NG-10-0110GC^(guan)T AT^(guan)T ACC T^(guan)TA ACC CAG NG-10-0323GC^(pyr)T AT^(pyr)T ACC T^(pyr)TA ACC CAG PMOplus; GC+T AT+T ACC +TTA ACC CAG NG-10-0301 NG-10-0248GCaT AaTaT ACC aTaTA ACC CAG NG-10-0600 * GCaT ATaT ACC TaTA ACC CAGNG-10-0602 ** GCpT ATpT ACC TpTA ACC CAG NG-10-0389GCX ATX ACC TXA ACC CAG NG-10-0247 GCpT ApTpT ACC TpTA ACC CAGNG-10-0299 GCaT ATaT ACC TaTA ACC CAG NG-10-0355 ***GCaT ATaT ACC TaTA ACC CAG * trimethyl glycine acylated product fromNG-10-0299; ** pT = PMOpyr methylated to quaternary amine fromNG-10-0323; X = PMOapn; *** 3′ trityl

Example 29 Treatment of Influenza A Virus Infected Cells with ExemplaryPMO Oligomers of the Invention

A series of PMO containing various modified intersubunit linkages wasprepared and used to treat influenza A virus-infected cells in culture.The PMO and PMO containing the modified intersubunit linkages of thepresent inventions were all designed to target the viral M1/M2 segmentat the AUG start codon and have one of two base sequences (SEQ ID NOs: 3and 4). PMO with the modified intersubunit linkages of the presentinvention are listed in Table 4 and identified by the NG numberdesignation in the Sequence Listing Table below. Inhibition of influenzaA virus replication by antisense targeting of multiple sites within theM1/M2 segment is described in co-owned and co-pending U.S. applicationSer. No. 12/945,081 which is incorporated herein by reference in itsentirety. In addition to inhibition of translation by targeting thecommon M1/M2 AUG start site, splice donor and splice acceptor sites canalso be targeted using compounds of the invention.

An alveolar murine macrophage cell line (ATCC; AMJ2-C11) was infected at0.1 MOI with H1N1 (strain PR8) and 1 hour post-infection PMO were added.Cells were incubated at 35 degrees C. overnight. Viral supernatant wasthen taken and incubated with VNAR protease to release viral RNA. HA RNAwas quantified by quantitative real-time PCR (qRT-PCR). Cells werewashed, fixed, and permeabilized. M1 and M2 proteins were then probedwith monoclonal antibodies for 30 min at 37 degrees C. Cells were washedand anti-mouse IgG conjugated with Alexa 646 was added for 15 min atroom temperature. M1 and M2 were then assayed by flow cytometry. Todetermine M1 and M2 protein levels, the percent of M1 or M2 positivecells was multiplied by the mean flourescent intensity of M1 or M2. Eachsample was then divided by the untreated control to generate the percentof M1 or M2 compared to untreated scramble controls.

FIG. 8 shows the reduction in viral M2 protein levels from cells treatedwith various compounds of the disclosure. The flow cytometry methoddescribed above was used to determine relative M2 protein expressionafter treatment at 60 micromolar. The oligomers inhibited the productionof the M2 protein to varying degrees with NG-10-180 (SEQ ID NO: 3)containing linkage b1 being the most active. Results using PMO withoutany intersubunit modifications is shown in FIG. 8 as NG-10-0015 (SEQ IDNO:3) for comparison.

Example 30 Treatment of Influenza A Virus Infected Mice In Vivo withExemplary PMO Oligomers of the Invention

Additional experiments in support of the invention were performed usingBalb/c mice infected with the PR8 strain of influenza A. Mice wereinfected with 3.5 TCID₅₀ via an intranasal inoculation after beingtreated 4 hours prior with PMO-X compounds of the invention. In someexperiments an additional dose of PMO-X was administered at 96 hrpost-infection. All doses consisted of 100 micrograms of test compoundin 50 microliters of PBS and were administered by intranasalinsufflation. The weight of the animals were monitored daily and wasused as a clinical endpoint for antiviral drug activity. At day 7post-infection the animals were sacrificed and lungs were harvested forviral load determinations using the qRT-PCR method described above inExample 29.

TCID₅₀ determinations were made using half-log serial dilutions of thelung homogenates and plated onto AMJ-C12 macrophage cells. After 24 hrat 35 degrees C., the media was changed and incubated for an additional72 h at 35 degrees C. 50 mL of a solution of 0.5% chicken RBC in PBS wasadded and incubated for 1 h at 4 degrees C. Hemagglutination pattern wasread and TCID₅₀ were calculated using the Reed and Muench method. TCID50values were then normalized to input tissue weight.

As shown in FIG. 9, PMO-X compounds show increased antiviral activityand decreased weight loss compared to a PMOplus compound after H1N1infection. Balb/c mice (n=4) were infected with H1N1 and given a single100 microgram dose of PMO 4 hours prior to infection. Mice were weigheddaily and percent weight loss was determined from pre-infection weight.Lungs were harvested day 7 post-infection and assayed for viral load byTCID₅₀. Results are presented as the fold increase in antiviral activityover naked PMO. This experiment shows approximately 50-fold increasedantiviral activity of two PMO-X compounds (NG-10-0097 and NG-11-0173;SEQ ID NO:3) compared to un-modified PMO (NG-10-0015; SEQ ID NO: 3) andapproximately 10-fold higher activity compared to a PMOplus compound(NG-11-0170; SEQ ID NO: 3).

FIG. 10 shows a similar experiment to that described for FIG. 9 usingbody weight as a clinical measurement of antiviral activity. Relative tothe PMOplus compound (NG-11-0170) several PMO-X compounds showedsuperior results including compounds containing succinoyl (NG-10-0108),isopropyl piperazine (NG-11-0148) and pyrollidone (NG-11-0173) linkagesand a PMOplus compound modified with a 3′ terminal benzhydryl group(NG-11-0145).

Example 31 Preparation of an Oligonucleotide Analogue Comprising aModified Terminal Group

To a solution of a 25-mer PMO containing a free 3′-end (27.7 mg, 3.226μmol) in DMSO (3004) was added farnesyl bromide(1.75 μl, 6.452 μmol) anddiisopropylethylamine (2.24 μL, 12.9 mop. The reaction mixture wasstirred at room temperature for 5 hours. The crude reaction mixture wasdiluted with 10 mL of 1% aqueous NH₄OH, and then loaded onto a 2 mLAmberchrome CG300M column. The column was then rinsed with 3 columnvolumes of water, and the product was eluted with 6 mL of 1:1acetonitrile and water (v/v). The solution was then lyophilized toobtain the title compound as a white solid.

Example 32 Preparation of Morpholino Oligomers

Preparation of trityl piperazine phenyl carbamate 35 (see FIG. 3): To acooled suspension of compound 11 in dichloromethane (6 mL/g 11) wasadded a solution of potassium carbonate (3.2 eq) in water (4 mL/gpotassium carbonate). To this two-phase mixture was slowly added asolution of phenyl chloroformate (1.03 eq) in dichloromethane (2 g/gphenyl chloroformate). The reaction mixture was warmed to 20° C. Uponreaction completion (1-2 hr), the layers were separated. The organiclayer was washed with water, and dried over anhydrous potassiumcarbonate. The product 35 was isolated by crystallization fromacetonitrile. Yield=80%

Preparation of carbamate alcohol 36: Sodium hydride (1.2 eq) wassuspended in 1-methyl-2-pyrrolidinone (32 mL/g sodium hydride). To thissuspension were added triethylene glycol (10.0 eq) and compound 35 (1.0eq). The resulting slurry was heated to 95° C. Upon reaction completion(1-2 hr), the mixture was cooled to 20° C. To this mixture was added 30%dichloromethane/methyl tert-butyl ether (v:v) and water. Theproduct-containing organic layer was washed successively with aqueousNaOH, aqueous succinic acid, and saturated aqueous sodium chloride. Theproduct 36 was isolated by crystallization from dichloromethane/methyltert-butyl ether/heptane. Yield=90%.

Preparation of Tail acid 37: To a solution of compound 36 intetrahydrofuran (7 mL/g 36) was added succinic anhydride (2.0 eq) andDMAP (0.5 eq). The mixture was heated to 50° C. Upon reaction completion(5 hr), the mixture was cooled to 20° C. and adjusted to pH 8.5 withaqueous NaHCO3. Methyl tert-butyl ether was added, and the product wasextracted into the aqueous layer. Dichloromethane was added, and themixture was adjusted to pH 3 with aqueous citric acid. Theproduct-containing organic layer was washed with a mixture of pH=3citrate buffer and saturated aqueous sodium chloride. Thisdichloromethane solution of 37 was used without isolation in thepreparation of compound 38.

Preparation of 38: To the solution of compound 37 was addedN-hydroxy-5-norbornene-2,3-dicarboxylic acid imide (HONB) (1.02 eq),4-dimethylaminopyridine (DMAP) (0.34 eq), and then1-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) (1.1eq). The mixture was heated to 55° C. Upon reaction completion (4-5 hr),the mixture was cooled to 20° C. and washed successively with 1:1 0.2 Mcitric acid/brine and brine. The dichloromethane solution underwentsolvent exchange to acetone and then to N,N-dimethylformamide, and theproduct was isolated by precipitation from acetone/N,N-dimethylformamideinto saturated aqueous sodium chloride. The crude product was reslurriedseveral times in water to remove residual N,N-dimethylformamide andsalts. Yield=70% of 38 from compound 36. Introduction of the activated“Tail” onto the disulfide anchor-resin was performed in NMP by theprocedure used for incorporation of the subunits during solid phasesynthesis.

Preparation of the Solid Support for Synthesis of Morpholino Oligomers:This procedure was performed in a silanized, jacketed peptide vessel(custom made by ChemGlass, NJ, USA) with a coarse porosity (40-60 μm)glass frit, overhead stirrer, and 3-way Teflon stopcock to allow N2 tobubble up through the frit or a vacuum extraction. Temperature controlwas achieved in the reaction vessel by a circulating water bath.

The resin treatment/wash steps in the following procedure consist of twobasic operations: resin fluidization and solvent/solution extraction.For resin fluidization, the stopcock was positioned to allow N2 flow upthrough the frit and the specified resin treatment/wash was added to thereactor and allowed to permeate and completely wet the resin. Mixing wasthen started and the resin slurry mixed for the specified time. Forsolvent/solution extraction, mixing and N2 flow were stopped and thevacuum pump was started and then the stopcock was positioned to allowevacuation of resin treatment/wash to waste. All resin treatment/washvolumes were 15 mL/g of resin unless noted otherwise.

To aminomethylpolystyrene resin (100-200 mesh; ˜1.0 mmol/g N2substitution; 75 g, 1 eq, Polymer Labs, UK, part #1464-X799) in asilanized, jacketed peptide vessel was added 1-methyl-2-pyrrolidinone(NMP; 20 ml/g resin) and the resin was allowed to swell with mixing for1-2 hr. Following evacuation of the swell solvent, the resin was washedwith dichloromethane (2×1-2 min), 5% diisopropylethylamine in 25%isopropanol/dichloromethane (2×3-4 min) and dichloromethane (2×1-2 min).After evacuation of the final wash, the resin was fluidized with asolution of disulfide anchor 34 in 1-methyl-2-pyrrolidinone (0.17 M; 15mL/g resin, ˜2.5 eq) and the resin/reagent mixture was heated at 45° C.for 60 hr. On reaction completion, heating was discontinued and theanchor solution was evacuated and the resin washed with1-methyl-2-pyrrolidinone (4×3-4 min) and dichloromethane (6×1-2 min).The resin was treated with a solution of 10% (v/v) diethyl dicarbonatein dichloromethane (16 mL/g; 2×5-6 min) and then washed withdichloromethane (6×1-2 min). The resin 39 (see FIG. 4) was dried under aN2 stream for 1-3 hr and then under vacuum to constant weight (±2%).Yield: 110-150% of the original resin weight.

Determination of the Loading of Aminomethylpolystyrene-disulfide resin:The loading of the resin (number of potentially available reactivesites) is determined by a spectrometric assay for the number oftriphenylmethyl (trityl) groups per gram of resin.

A known weight of dried resin (25±3 mg) is transferred to a silanized 25ml volumetric flask and ˜5 mL of 2% (v/v) trifluoroacetic acid indichloromethane is added. The contents are mixed by gentle swirling andthen allowed to stand for 30 min. The volume is brought up to 25 mL withadditional 2% (v/v) trifluoroacetic acid in dichloromethane and thecontents thoroughly mixed. Using a positive displacement pipette, analiquot of the trityl-containing solution (500 μL) is transferred to a10 mL volumetric flask and the volume brought up to 10 mL withmethanesulfonic acid.

The trityl cation content in the final solution is measured by UVabsorbance at 431.7 nm and the resin loading calculated in trityl groupsper gram resin (μmol/g) using the appropriate volumes, dilutions,extinction coefficient (ε: 41 μmol-1 cm-1) and resin weight. The assayis performed in triplicate and an average loading calculated.

The resin loading procedure in this example will provide resin with aloading of approximately 500 μmol/g. A loading of 300-400 in μmol/g wasobtained if the disulfide anchor incorporation step is performed for 24hr at room temperature.

Tail loading: Using the same setup and volumes as for the preparation ofaminomethylpolystyrene-disulfide resin, the Tail can be introduced intothe molecule. For the coupling step, a solution of 38 (0.2 M) in NMPcontaining 4-ethylmorpholine (NEM, 0.4 M) was used instead of thedisulfide anchor solution. After 2 hr at 45° C., the resin 39 was washedtwice with 5% diisopropylethylamine in 25% isopropanol/dichloromethaneand once with DCM. To the resin was added a solution of benzoicanhydride (0.4 M) and NEM (0.4 M). After 25 min, the reactor jacket wascooled to room temperature, and the resin washed twice with 5%diisopropylethylamine in 25% isopropanol/dichloromethane and eight timeswith DCM. The resin 40 was filtered and dried under high vacuum. Theloading for resin 40 is defined to be the loading of the originalaminomethylpolystyrene-disulfide resin 39 used in the Tail loading.

Solid Phase Synthesis: Morpholino Oligomers were prepared on a GilsonAMS-422 Automated Peptide Synthesizer in 2 mL Gilson polypropylenereaction columns (Part #3980270). An aluminum block with channels forwater flow was placed around the columns as they sat on the synthesizer.The AMS-422 will alternatively add reagent/wash solutions, hold for aspecified time, and evacuate the columns using vacuum.

For oligomers in the range up to about 25 subunits in length,aminomethylpolystyrene-disulfide resin with loading near 500 μmol/g ofresin is preferred. For larger oligomers,aminomethylpolystyrene-disulfide resin with loading of 300-400 μmol/g ofresin is preferred. If a molecule with 5′-Tail is desired, resin thathas been loaded with Tail is chosen with the same loading guidelines.

The following reagent solutions were prepared:

Detritylation Solution: 10% Cyanoacetic Acid (w/v) in 4:1dichloromethane/acetonitrile; Neutralization Solution: 5%Diisopropylethylamine in 3:1 dichloromethane/isopropanol; CouplingSolution: 0.18 M (or 0.24 M for oligomers having grown longer than 20subunits) activated Morpholino Subunit of the desired base and linkagetype and 0.4 M N ethylmorpholine, in 1,3-dimethylimidazolidinone.Dichloromethane (DCM) was used as a transitional wash separating thedifferent reagent solution washes.

On the synthesizer, with the block set to 42° C., to each columncontaining 30 mg of aminomethylpolystyrene-disulfide resin (or Tailresin) was added 2 mL of 1-methyl-2-pyrrolidinone and allowed to sit atroom temperature for 30 min. After washing with 2 times 2 mL ofdichloromethane, the following synthesis cycle was employed:

Step Volume Delivery Hold time Detritylation 1.5 mL Manifold 15 secondsDetritylation 1.5 mL Manifold 15 seconds Detritylation 1.5 mL Manifold15 seconds Detritylation 1.5 mL Manifold 15 seconds Detritylation 1.5 mLManifold 15 seconds Detritylation 1.5 mL Manifold 15 secondsDetritylation 1.5 mL Manifold 15 seconds DCM 1.5 mL Manifold 30 secondsNeutralization 1.5 mL Manifold 30 seconds Neutralization 1.5 mL Manifold30 seconds Neutralization 1.5 mL Manifold 30 seconds Neutralization 1.5mL Manifold 30 seconds Neutralization 1.5 mL Manifold 30 secondsNeutralization 1.5 mL Manifold 30 seconds DCM 1.5 mL Manifold 30 secondsCoupling 350 uL-500 uL Syringe 40 minutes DCM 1.5 mL Manifold 30 secondsNeutralization 1.5 mL Manifold 30 seconds Neutralization 1.5 mL Manifold30 seconds DCM 1.5 mL Manifold 30 seconds DCM 1.5 mL Manifold 30 secondsDCM 1.5 mL Manifold 30 seconds

The sequences of the individual oligomers were programmed into thesynthesizer so that each column receives the proper coupling solution(A,C,G,T,I) in the proper sequence. When the oligomer in a column hadcompleted incorporation of its final subunit, the column was removedfrom the block and a final cycle performed manually with a couplingsolution comprised of 4-methoxytriphenylmethyl chloride (0.32 M in DMI)containing 0.89 M 4-ethylmorpholine.

Cleavage from the resin and removal of bases and backbone protectinggroups: After methoxytritylation, the resin was washed 8 times with 2 mL1-methyl-2-pyrrolidinone. One mL of a cleavage solution consisting of0.1 M 1,4-dithiothreitol (DTT) and 0.73 M triethylamine in1-methyl-2-pyrrolidinone was added, the column capped, and allowed tosit at room temperature for 30 min. After that time, the solution wasdrained into a 12 mL Wheaton vial. The greatly shrunken resin was washedtwice with 300 μL of cleavage solution. To the solution was added 4.0 mLconc aqueous ammonia (stored at −20° C.), the vial capped tightly (withTeflon lined screw cap), and the mixture swirled to mix the solution.The vial was placed in a 45° C. oven for 16-24 hr to effect cleavage ofbase and backbone protecting groups.

Initial Oligomer Isolation: The vialed ammonolysis solution was removedfrom the oven and allowed to cool to room temperature. The solution wasdiluted with 20 mL of 0.28% aqueous ammonia and passed through a 2.5×10cm column containing Macroprep HQ resin (BioRad). A salt gradient (A:0.28% ammonia with B: 1 M sodium chloride in 0.28% ammonia; 0-100% B in60 min) was used to elute the methoxytrityl containing peak. Thecombined fractions were pooled and further processed depending on thedesired product.

Demethoxytritylation of Morpholino Oligomers: The pooled fractions fromthe Macroprep purification were treated with 1 M H3PO4 to lower the pHto 2.5. After initial mixing, the samples sat at room temperature for 4min, at which time they are neutralized to pH 10-11 with 2.8%ammonia/water. The products were purified by solid phase extraction(SPE).

Amberchrome CG-300M (Rohm and Haas; Philadelphia, Pa.) (3 mL) is packedinto 20 mL fitted columns (BioRad Econo-Pac Chromatography Columns(732-1011)) and the resin rinsed with 3 mL of the following: 0.28%NH4OH/80% acetonitrile; 0.5M NaOH/20% ethanol; water; 50 mM H3PO4/80%acetonitrile; water; 0.5 NaOH/20% ethanol; water; 0.28% NH4OH.

The solution from the demethoxytritylation was loaded onto the columnand the resin rinsed three times with 3-6 mL 0.28% aqueous ammonia. AWheaton vial (12 mL) was placed under the column and the product elutedby two washes with 2 mL of 45% acetonitrile in 0.28% aqueous ammonia.The solutions were frozen in dry ice and the vials placed in a freezedryer to produce a fluffy white powder. The samples were dissolved inwater, filtered through a 0.22 micron filter (Pall Life Sciences,Acrodisc 25 mm syringe filter, with a 0.2 micron HT Tuffryn membrane)using a syringe and the Optical Density (OD) was measured on a UVspectrophotometer to determine the OD units of oligomer present, as wellas dispense sample for analysis. The solutions were then placed back inWheaton vials for lyophilization.

Analysis of Morpholino Oligomers: MALDI-TOF mass spectrometry was usedto determine the composition of fractions in purifications as well asprovide evidence for identity (molecular weight) of the oligomers.Samples were run following dilution with solution of3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid),3,4,5-trihydoxyacetophenone (THAP) or alpha-cyano-4-hydoxycinnamic acid(HCCA) as matrices.

Cation exchange (SCX) HPLC was performed using a Dionex ProPac SCX-10,4×250 mm column (Dionex Corporation; Sunnyvale, Calif.) using 25 mM pH=5sodium acetate 25% acetonitrile (Buffer A) and 25 mM pH=5 sodium acetate25% acetonitrile 1.5 M potassium chloride (buffer B) (Gradient 10-100% Bin 15 min) or 25 mM KH2PO4 25% acetonitrile at pH=3.5 (buffer A) and 25mM KH2PO4 25% acetonitrile at pH=3.5 with 1.5 M potassium chloride(buffer B) (Gradient 0-35% B in 15 min). The former system was used forpositively charged oligomers that do not have a peptide attached, whilethe latter was used for peptide conjugates.

Purification of Morpholino Oligomers by Cation Exchange Chromatography:The sample is dissolved in 20 mM sodium acetate, pH=4.5 (buffer A) andapplied to a column of Source 30 cation exchange resin (GE Healthcare)and eluted with a gradient of 0.5 M sodium chloride in 20 mM sodiumacetate and 40% acetonitrile, pH=4.5 (buffer B). The pooled fractionscontaining product are neutralized with conc aqueous ammonia and appliedto an Amberchrome SPE column. The product is eluted, frozen, andlyophilized as above.

TABLE 11 Sequence Listing SEQ Name Sequence (5′ to 3′) ID NO DengueCGGTCCACGTAGACTAACAACT  1 JEV GAAGTTCACACAGATAAACTTCT  2 M1/M2AUG.20.22CGGTTAGAAGACTCATCTTT  3 M1/M2AUG.25.26 TTTCGACATCGGTTAGAAGACTCAT  4NP-AUG GAGACGCCATGATGTGGATGTC  5 Picornavirus GAAACACGGACACCCAAAGTAGT  6Dengue 3′-CS TCCCAGCGTCAATATGCTGTTT  7 Arenaviruses GCCTAGGATCCACGGTGCGC 8 RSV-L target GGGACAAAATGGATCCCATTATTA  9 ATGGAAATTCTGCTAA RSV-AUG-2TAATGGGATCCATTTTGTCCC 10 RSV-AUG3 AATAATGGGATCCATTTTGTCCC 11 RSV-AUG4CATTAATAATGGGATCCATTTTGTCCC 12 RSV-AUG5 GAATTTCCATTAATAATGGGATCCATTTTG13 RSV-AUG6 CAGAATTTCCATTAATAATGGGATCCATT 14 M23DGGCCAAACCTCGGCTTACCTGAAAT 15 AVI-5225 GGCCAAACCTCGGCTTACCTGAAAT- 16/79RAhxRRBRRAhxRRBRAhxB eGFP654 GCTATTACCTTAACCCAG 17 huMSTN targetGAAAAAAGATTATATTGATTTTAAAATCAT 18 GCAAAAACTGCAACTCTGTGTT muMSTN25-104CATACATTTGCAGTTTTTGCATCAT 19 muMSTN25-183 TCATTTTTAAAAATCAGCACAATCTT 20muMSTN25-194 CAGTTTTTGCATCATTTTTAAAAATC 21 Exon44-AGATCTGTCAAATCGCCTGCAGGTAA 22 Exon44-B AAACTGTTCAGCTTCTGTTAGCCAC 23Exon44-C TTGTGTCTTTCTGAGAAACTGTTCA 24 Exon45-A CTGACAACAGTTTGCCGCTGCCCAA25 Exon45-B CCAATGCCATCCTGGAGTTCCTGTAA 26 Exon45-CCATTCAATGTTCTGACAACAGTTTGCCGCT 27 Exon50-A CTTACAGGCTCCAATAGTGGTCAGT 28Exon50-B CCACTCAGAGCTCAGATCTTCTAACTTCC 29 Exon50-CGGGATCCAGTATACTTACAGGCTCC 30 Exon51-A ACATCAAGGAAGATGGCATTTCTAGTTTGG 31Exon51-B CTCCAACATCAAGGAAGATGGCATTTCTAG 32 Exon51-CGAGCAGGTACCTCCAACATCAAGGAA 33 Exon53-A CTGAAGGTGTTCTTGTACTTCATCC 34Exon53-B TGTTCTTGTACTTCATCCCACTGATTCTGA 35 SMN2-A CTTTCATAATGCTGGCAG 36SMN2-B CATAATGCTGGCAG 37 SMN2-C GCTGGCAG 38 CAG 9mer CAG CAG CAG 39CAG 12mer CAG CAG CAG CAG 40 CAG 15mer CAG CAG CAG CAG CAG 41 CAG 18merCAG CAG CAG CAG CAG CAG 42 AGC 9mer AGC AGC AGC 43 AGC 12merAGC AGC AGC AGC 44 AGC 15mer AGC AGC AGC AGC AGC 45 AGC 18merAGC AGC AGC AGC AGC AGC 46 GCA 9mer GCA GCA GCA 47 GCA 12merGCA GCA GCA GCA 48 GCA 15mer GCA GCA GCA GCA GCA 49 GCA 18merGCA GCA GCA GCA GCA GCA 50 AGC 25mer AGC AGC AGC AGC AGC AGC 51AGC AGC A CAG 25mer CAG CAG CAG CAG CAG CAG 52 CAG CAG C CAGG 9merCAG GCA GGC 53 CAGG 12mer CAG GCA GGC AGG 54 CAGG 24merCAG GCA GGC AGG CAG GCA 55 GGC AGGArginine-Rich Cell Penetrating Peptides rTAT RRRQRRKKR 56 Tat RKKRRQRRR57 R₉F₂ RRRRRRRRRFF 58 R₅F₂R₄ RRRRRFFRRRR 59 R₄ RRRR 60 R₅ RRRRR 61 R₆RRRRRR 62 R₇ RRRRRRR 63 R₈ RRRRRRRR 64 R₉ RRRRRRRRR 65 (RAhxR)₄;RAhxRRAhxRRAhxRRAhxR 66 (P007) (RAhxR)₅; RAhxRRAhxRRAhxRRAhxRRAhxR 67(CP04057) (RAhxRRBR)₂; RAhxRRBRRAhxRRBR 68 (CP06062) (RAR)₄F₂RARRARRARRARFFC 69 (RGR)₄F₂ RGRRGRRGRRGRFFC 70

The various embodiments described above can be combined to providefurther embodiments. All of the U.S. patents, U.S. patent applicationpublications, U.S. patent applications, foreign patents, foreign patentapplications and non-patent publications referred to in thisspecification and/or listed in the Application Data Sheet, areincorporated herein by reference, in their entirety. Aspects of theembodiments can be modified, if necessary to employ concepts of thevarious patents, applications and publications to provide yet furtherembodiments. These and other changes can be made to the embodiments inlight of the above-detailed description. In general, in the followingclaims, the terms used should not be construed to limit the claims tothe specific embodiments disclosed in the specification and the claims,but should be construed to include all possible embodiments along withthe full scope of equivalents to which such claims are entitled.Accordingly, the claims are not limited by the disclosure.

1.-80. (canceled)
 81. A morpholino subunit, wherein the morpholinosubunit has the following structure (XXXI)

or a salt or isomer thereof, wherein: W is, at each occurrence,independently S or O; X is, at each occurrence, independently —NR⁸R⁹ or—OR³; and Y is, at each occurrence, independently O or —NR¹⁰, R⁸ is, ateach occurrence, independently hydrogen or C₂-C₁₂ alkyl; R⁹ is, at eachoccurrence, independently hydrogen, C₁-C₁₂ alkyl, C₁-C₁₂ aralkyl oraryl; R¹⁰ is, at each occurrence, independently hydrogen, C₁-C₁₂ alkylor -LNR⁴R⁵R⁷; wherein R⁸ and R⁹ may join to form a 5-18 membered mono orbicyclic heterocycle or R⁸, R⁹ or R³ may join with R¹⁰ to form a 5-7membered heterocycle, and wherein when X is 4-piparazino, X has thefollowing structure (III):

wherein: R¹¹ is, at each occurrence, independently C₂-C₁₂ alkyl, C₁-C₁₂aminoalkyl, C₁-C₁₂ alkylcarbonyl, aryl, heteroaryl or heterocyclyl; andR is, at each occurrence, independently an electron pair, hydrogen orC₁-C₁₂ alkyl; and R¹² is, at each occurrence, independently, hydrogen,C₁-C₁₂ alkyl, C₁-C₁₂ aminoalkyl, —NH₂, —CONH₂, —NR¹³R¹⁴, —NR¹³R¹⁴R¹⁵,C₁-C₁₂ alkylcarbonyl, oxo, —CN, trifluoromethyl, amidyl, amidinyl,amidinylalkyl, amidinylalkylcarbonyl guanidinyl, guanidinylalkyl,guanidinylalkylcarbonyl, cholate, deoxycholate, aryl, heteroaryl,heterocycle, —SR¹³ or C₁-C₁₂ alkoxy, wherein R¹³, R¹⁴ and R¹⁵ are, ateach occurrence, independently C₁-C₁₂ alkyl; and wherein at least one ofthe intersubunit linkages is linkage (B) Z is halo or a linkage to asolid support; and PG is C₇-C₃₀ aralkyl.
 82. The morpholino subunit ofclaim 81, wherein Z is chloro.
 83. The morpholino subunit of claim 81,wherein PG is trityl or methoxy trityl.